Gene expression system using stealthy rna, and gene introduction/expression vector including said rna

ABSTRACT

Simultaneous expression of a plurality of foreign genes by using a stealthy RNA gene expression system that is a complex that does not activate the innate immune mechanism and is formed from an RNA-dependent RNA polymerase, a single-strand RNA binding protein, and negative-sense single-strand RNAs including the following (1) to (8): (1) a target RNA sequence that codes for any protein or functional RNA; (2) an RNA sequence forming a noncoding region and derived from mRNA; (3) a transcription initiation signal sequence recognized by the RNA-dependent RNA polymerase; (4) a transcription termination signal sequence recognized by the polymerase; (5) an RNA sequence containing a replication origin recognized by the polymerase; (6) an RNA sequence that codes for the polymerase; (7) an RNA sequence that codes for a protein for regulating the activity of the polymerase; and (8) an RNA sequence that codes for the single-strand RNA binding protein.

TECHNICAL FIELD

The present invention relates to a vector for introducing andpersistently expressing exogenous genes in animal cells.

BACKGROUND ART

The techniques of externally introducing any given gene into animalcells including human cells, and expressing the gene persistently in thecells are essential techniques in various industries utilizingbiotechnologies. For example, industrial mass production of humanmonoclonal antibodies for use as pharmaceuticals requires the techniqueof persistently expressing genes of H-chain and L-chain ofimmunoglobulin at the same level. In gene therapy of congenitalmetabolic diseases, the technique of introducing a therapeutic gene intohuman tissue cells, and stably expressing the gene in the body for along term is required.

1. Regarding Cell-Reprogramming Technology

Recently, a cell-reprogramming technology for producing useful cells bygenetically converting the characteristic of normal tissue cellsattracts attention. The technique of introducing genes into an animalcell and persistently expressing the genes is also a base technologyessential for cell-reprogramming. For example, it is possible to preparehuman induced pluripotent stem cells (iPS cells) by introducing acombination of four genes, OCT4, SOX2, KLF4, and c-MYC, or OCT4, SOX2,NANOG, and LIN28 into human normal fibroblasts, and expressing the genespersistently for 21 days (Patent Document 1, Patent Document 2,Non-Patent Document 1, and Non-Patent Document 2). Also, it is possibleto prepare hepatic cells by introducing three genes, FOXA3, HNF1A, andHNF4A into human fibroblasts and expressing the gene persistently for 14days. (Non-Patent Document 3) It is also reported that a dopaminergicneuron can be prepared by introducing five genes, ASCL1, BRN2, MYT1L,LMX1A, and FOXA2 into human fibroblasts, and expressing the genepersistently for 24 days (Non-Patent Document 4). Thus, in variouscell-reprogramming, there is a need for a technique capable ofsimultaneously introducing and expressing plural genes into a cell, andkeeping the expression for a period required for reprogramming.

It is known that cell-reprogramming can be induced in vivo. For example,it has been reported that when three genes, GATA4, MEF2C, and TBX5, orfour genes, GATA4, HAND2, MEF2C, and TBX5 are administered to aninfarcted site in a mouse myocardial infarction model, infiltratedfibroblasts transdifferentiate into cardiomyocytes (Non-Patent Document5, and Non-Patent Document 6). Therefore, the cell-reprogrammingtechnology is expected to become the basis of regenerative medicine formyocardial infarction, spinal cord injury and the like in future.

2. Improvement in Cell-Reprogramming Efficiency

Assuming that vitro cell-reprogramming is used for medicine, it isdesired that the material cells can be collected from a human bodywithout invasion, and can be collected in the condition that they arenot contaminated with microorganisms outside the living body. Cells thatsatisfy these requirements are almost limited to mononuclear cells inperipheral blood, and a gene introduction vector adapted to these cellsis desired.

In general, the efficiency with which animal cells are reprogrammed byexternally introduced genes is very low, however, the efficiency can beraised by carrying all the genes on one vector, and introducing thegenes into cells at once (Patent Document 3, Patent Document 4,Non-Patent Document 7, and Non-Patent Document 8).

Also it is known that the efficiency is raised by increasing the numberof genes used in cell-reprogramming. For example, in the technique ofconverting mouse fibroblasts to induced pluripotent stem cells (iPScells), it is known that the efficiency of conversion rises five timesby using a total of six genes by adding two genes, BRG1 and BAF155, tofour genes, OCT4, SOX2, KLF4, and c-MYC (Non-Patent Document 9). Also,in the technique of reprogramming human fibroblasts into motor nerves,it is known that the efficiency of reprogramming rises 100 times byusing a total of seven genes by adding three genes, HB9, ISL1, and NGN2to four genes, LHX3, ASCL1, BRN2, and MYT1L (Non-Patent Document 10).

When the number of genes used in cell-reprogramming is increased, thesize of genes that should be carried also increases. Illustratingpreparation of iPS cells as an example, the total size of four genes,KLF4, OCT4, SOX2, and c-MYC is 4,774 base pairs, whereas the total sizeof genes after adding the two genes, BRG1 (5,040 base pairs) and BAF155(3,318 base pairs) is 13,132 base pairs (Non-Patent Document 9). Byadding CHD1 gene (5,133 base pairs) encoding a chromatin remodelingfactor that is specifically expressed in embryonic stem cells and isexpected to accelerate reprogramming of cells to iPS cells to fourgenes, KLF4, OCT4, SOX2, and c-MYC, the total size amounts to 9,907 basepairs, and by adding TET1 gene (6,429 base pairs) encoding a DNAdemethylase to four genes, KLF4, OCT4, SOX2, and c-MYC, the total sizeamounts to 11,203 base pairs. The total size of the seven genes, LHX3,ASCL1, BRN2, MYT1L, HB9, ISL1, and NGN2 that are used in the techniqueof reprogramming human fibroblasts into motor nerves is 9,887 base pairs(Non-Patent Document 10).

Thus, in order to raise the efficiency of the cell-reprogramming, it isdesired to use at least six or more genes, and a vector capable ofcarrying all of the genes at once is desired. Also, desired is a vectorcapable of expressing introduced exogenous genes even when the totalsize of the genes is 5,000 or more nucleotides, desirably 8,000 or morenucleotides.

The term vector used herein refers to a recombinant viral or non-viralnucleic acid-macromolecular substance complex that is composed ofnucleic acid including exogenous genes, and is capable of introducingthe nucleic acid into animal cells and expressing the genes.

It is known that in reprogramming of animal cells by expression ofexogenous genes, the expression levels of the genes seriously affect thecharacteristics of the reprogrammed cells. For example, when four genes,OCT4, SOX2, KLF4, and c-MYC are expressed in mouse fibroblasts, it isknown that iPS cells are generated when expression of the genes is weak,whereas cells having a totally different characteristic from iPS cellsare generated when the expression of the genes is strong (Non-PatentDocument 11). Thus, for the technique of reprogramming animal cellsincluding human cells by expressing externally introduced genes, thereis a need for a vector capable of setting the expression of the genes atan optimum level depending on the purpose.

3. Removal of Genes for Reprogramming

Further in order to make the reprogrammed cells prepared by externallyintroducing genes completely exert their function, it is necessary tocompletely remove the reprogramming genes from the cells. Also, when theprepared human cells are used as a material for regenerative medicine,it is necessary to completely remove the genes from the cells forensuring the safety. For example, in induced pluripotent stem cells (iPScells) prepared by using four genes, OCT4,SOX2, KLF4, and c-MYC, thepluripotency cannot be functional in the condition that these four genesare expressed, and hence, it is necessary to at least completelysuppress the expression of these genes, or preferably completely removethese genes from the cells (Patent Document 1, Patent Document 2, PatentDocument 3, Non-Patent Document 1, Non-Patent Document 2 and Non-PatentDocument 7). It is also known that if the c-MYC gene used in preparingthe iPS cells is left in the iPS cells, the tissue cells that areprepared by differentiation of the iPS cells become tumorigenic withhigh frequency (Non-Patent Document 12). Therefore, it is necessary tocompletely remove the c-MYC gene from the iPS cells for ensuring thesafety.

Thus, the gene expression technique required for cell-reprogrammingneeds to have the mutually contradictory characteristics: persistentexpression of genes at an optimum levels is desired for achieving thereprogramming, while it can be removed easily and completely once thereprogramming has completed.

4. Importance of Avoiding Activation of Innate Immune System

Most of the gene introduction/expression vectors that are currently usedin animal cells are constructed using an animal viruses or plasmid DNAsprepared from microorganisms such as Escherichia coli as materials.However, an animal cell has an innate immune system that eliminatesinvading pathogens from outside (Non-Patent Document 13) and nucleicacids derived from viruses or microorganisms introduced from outside thecell are recognized as foreign substances, and the innate immune systemis activated. When the degree activation of the innate immune systemexceeds a certain level, cell death by the apoptosis is induced, andthus the efficiency of the reprogramming is deteriorated. Whenexpression of interferon or inflammatory cytokines is induced by theactivation of the innate immune system, inflammation is caused in theliving body. In order to prevent such an undesired reaction, geneintroduction/expression technique for cell-reprogramming is required tobe capable of avoiding the activation of the innate immune system. Thischaracteristic is important particularly in application to theregenerative medicine including in vivo cell-reprogramming as describedin the above section 1.

5. Gene Introduction/Expression System for Ideal Cell-Reprogramming

From the foregoing investigation, there is a need for a geneintroduction/expression technique satisfying the following at least fiverequirements as discussed in the above sections 1 to 4. so as to furtherameliorate the cell-reprogramming technique for animal cells includinghuman cells by using genes for industrial application.

(1) Capability of efficiently introducing exogenous genes into animalcells including human peripheral blood cells.

(2) Capability of persistently expressing the genes for any requiredperiod.

(3) Capability of avoiding the innate immune system possessed by cellsin expression of the genes.

(4) Capability of expressing the genes even if the total length of theintroduced exogenous genes is 5,000 or more nucleotides, desirably 8,000or more nucleotides.

(5) Capability of simultaneously expressing at least six, desirablyeight or more genes.

Also, it is greatly desired to further achieve the following points.

(6) Capability of regulating the expression levels of the genes. Inparticular, it is preferred that the expression level of each gene canbe regulated individually when plural genes are introduced.

In applying gene-introduced cells, in particular, to transplantationtechniques, the following point is also very important.

(7) Capability of removing the gene by a simple technique when the genesbecome unnecessary.

6. Technique of Introducing Plural Genes Into Animal Cells

As a technique for introducing plural genes into animal cells includinghuman cells from outside, and expressing the genes persistently in thecells, that has been reported to be applicable to cell-reprogramming,the following three techniques are known.

(1) Method of integrating the genes into nuclear genomic DNA.

(2) Method of carrying the genes on DNA capable of existing stably andindependently from genomic DNA in a nucleus.

(3) Method of carrying the genes on RNA capable of existing incytoplasm.

6-1. Method for Integrating Plural Genes Into Nuclear Genomic DNA

In the method of integrating an exogenous gene into genomic DNA existingin a nucleus of cell by using a lentivirus vector (Non-Patent Document8, and Non-Patent Document 14), transposon (Non-Patent Document 15, andNon-Patent Document 16), non-homologous recombination, homologousrecombination or the like, the gene can exist stably as with the genomicDNA. However, once the gene is integrated into the genomic DNA,complicated operations such as introducing a sequence specificrecombinase into cells are required for selectively removing the genefrom the genomic DNA, and the gene cannot be removed securely from everycell (Non-Patent Document 15). Further, since integration of exogenousgenes into genomic DNA requires DNA replication of host cells, theefficiency of gene introduction into cells having poor proliferationpotency such as blood cells is very low. Further, the phenomenon of“insertional mutagenesis” that random integration of exogenous gene intogenomic DNA causes disruption or abnormal activation of genes of thehost is known, and hence, there exists a concern about the safety formedical application (Non-Patent Document 17).

6-2. Method for Carrying Plural Genes on a DNA that is Independent fromGenomic DNA in Nucleus

As a method for carrying a exogenous gene on a DNA capable of existingstably in a nucleus of cell independently from genomic DNA, a method ofusing a circular DNA carrying a replication origin of genome ofEpstein-Barr virus (Non-Patent Document 18), and a method of using anartificial chromosome containing a straight-chain giant DNA (Non-PatentDocument 19) are known. These DNA molecules continue replication and arekept stably in nuclei of human cells, and the mechanism of this relieson the mechanism with which genomic DNA of host cells is replicated.Therefore, it is impossible to specifically inhibit only replication ofthe DNA carrying exogenous genes, and a technique for actively removingthe DNA from cells has not been reported. Additionally, since divisionof a host cell is required for introducing the DNA molecule into a cellnucleus, the efficiency of gene introduction into cells having poorproliferation potency such as blood cells is very low. Further, since itis known that circular DNA in a cell nucleus is frequently incorporatedinto genomic DNA of the cell, the risk of insertional mutagenesis cannotbe eliminated (Non-Patent Document 20).

6-3. Technique of Expressing Plural Genes from Single Vector DNA

Further, as described in the above sections 6-1. and 6-2., when DNA isused as a platform for gene expression, a technique of expressing pluralgenes from the single vector DNA is required. As such a technique, thefollowing three methods are known: 1) a method of simply linking pluralindependent genes, and expressing the genes, 2) a method of expressingplural proteins from one messenger RNA (mRNA) by using an RNA structurecalled Internal Ribosome Entry Site (IRES), and a method of expressing afusion protein in which plural proteins are linked by 2A peptide.

It is known that in the method of linking plural independent genes,expression of genes is strongly suppressed due to mutual interferencebetween genes (Non-Patent Document 21). In order to prevent this, it isnecessary to insert a structure called an insulator between genes, andthe insertion increases the size of the vector DNA, and complicates thestructure of the vector DNA. While the case of expressing four genesinstalled on one DNA molecule has been reported in this method(Non-Patent Document 22), the case of simultaneously expressing five ormore genes has not been reported.

In the method of expressing plural proteins from one messenger RNA(mRNA) by using IRES sequence, the translation efficiency of the proteinpositioned downstream IRES sequence is lower than, or sometimes 10% orless compared with the translation efficiency of the protein positionedupstream IRES sequence (Non-Patent Document 23). Additionally, sinceIRES sequence has a relatively large size and has a complicatedstructure, the method of using IRES sequence is mainly used forsimultaneously expressing two proteins.

2A peptide has a structure consisting of 18 to 22 amino acid residuesfound in a positive-sense single-stranded RNA virus, and a fusionprotein in which plural proteins are connected by 2A peptide areautomatically cleaved at the time of synthesis and dissociated into theoriginal plural proteins. In this technique, one proline residue is leftat the N-terminus of each protein arising after cleavage, and 17 to 21amino acid residues are left at the C-terminus, and these excess aminoacid residues can influence on the function of the protein (Non-PatentDocument 24). In addition, since the efficiency of cleavage at a 2Apeptide site is largely influenced by the structure of the fusionprotein, it is necessary to make trial and error requiring labors forpreparing plural proteins efficiently (Non-Patent Document 25). In themethod of connecting plural proteins by 2A peptide, the case ofsimultaneously expressing four proteins (Non-Patent Document 8) and thecase of simultaneously expressing five proteins (Non-Patent Document 16)have been reported. Also the case of expressing four proteins bycombining IRES sequence and 2A peptide has been reported (Non-PatentDocument 4).

6-4. Method for Carrying Plural Genes on One RNA Existing in Cytoplasm

As described in the above sections 6-1. to 6-3., in the existing geneintroduction/expression technique that uses DNA as a platform for geneexpression, cell-reprogramming using four to five genes has beenreported. However, as long as DNA is used as a platform for geneexpression, it is not easy to simultaneously carry six or more genes andto achieve removal of the genes in a convenient way, and a techniquesatisfying at least all the five requirements required for idealreprogramming shown in the above section 5. has not been reported.

Meanwhile, as a technique of cell-reprogramming by expressing pluralgenes that are externally introduced into animal cells including humancells using RNA as a platform, techniques of using a positive-sense RNA(Non-Patent Document 26, and Non-Patent Document 27), and techniques ofusing a negative-sense RNA (Patent Document 3, Patent Document 4, PatentDocument 5, Patent Document 6, Non-Patent Document 7, Non-PatentDocument 28, Non-Patent Document 29, and Non-Patent Document 30) havebeen reported.

6-4-1. Method of Using Positive-Sense RNA

As a technique of cell-reprogramming by using a positive-sense RNAcapable of existing stably in cytoplasm, a technique of using apositive-sense single-stranded genomic RNA derived from Venezuelanequine encephalomyelitis virus (VEEV) (Non-Patent Document 26) has beenreported. In this technique, expression of four proteins is realized byreplacing a structural gene on 3′ side of genomic RNA of VEEV with genesencoding proteins that are linked by 2A peptide. This system inducesextremely strong expression of interferon, and then combination with ananti-interferon stance (B18R protein derived from vaccinia virus) isnecessarily required (Non-Patent Document 26). The efficiency of geneintroduction depends on the gene introducing reagent to be applied, andcells capable of being reprogrammed is limited to adhesive cells such asfibroblasts. An RNA carrying exogenous genes is unstable, and disappearsby removing B18R protein from the culture medium.

As a technique of cell reprogramming by using a positive-sense RNA, atechnique using a chemically synthesized messenger RNA (mRNA)(Non-Patent Document 27) has been reported. In this prior art, aftermixing plural mRNAs separately carrying up to five exogenous genes, theplural mRNAs are introduced into cells by using a gene introducingreagent. Since the expressions of the genes are transient, it isnecessary to newly introduce the genes into the cells every day. Also,the gene introduction is limited to adhesive cells such as fibroblasts.Also in this technique, since the innate immune system is activatedstrongly, it is necessary to combine an anti-interferon substance (B 18Rprotein derived from vaccinia virus) (Non-Patent Document 27).

6-4-2. Method of Using Negative-Sense RNA

As a technique of cell-reprogramming using negative-sense RNAs, a methodof using mixed vectors separately carrying an exogenous gene on awild-type strain of Sendai virus which is one species of paramyxoviruses(Patent Document 5, Non-Patent Document 28, and Non-Patent Document 29),and a method of using a vector carrying three genes simultaneously(Patent Document 6, and Non-Patent Document 30) have been reported asprior arts. In these gene expression systems using negative-senseRNA(s), autonomous replication ability of the wild-type virus isattenuated by deleting F gene, and exogenous genes are installedrespectively as single gene expression cassettes. Although activation ofthe innate immune system was not mentioned, the vectors are expected tohave ability to activate the innate immune system correspondinglybecause it has been known that Sendai virus which is a material hasstrong interferon inducibility (Non-Patent Document 31). Also it hasbeen reported that the vector can be removed by introducing atemperature sensitive mutation into genome of the wild-type virus, andthus increasing the cultivation temperature (Patent Document 6,Non-Patent Document 29, and Non-Patent Document 30). The size of genethat can be expressed by a vector based on wild-type Sendai virus hasbeen reported to be from 3078 base pairs (beta galactosidase fromEscherichia coli) (Non-Patent Document 32) to 3450 base pairs (sum ofthree genes, KLF4, OCT4, and SOX2) (Patent Document 6, Non-PatentDocument 30).

As technique of reprogramming cells by using a negative sense RNA, atechnique based on a mutant Sendai virus capably of persistent infectionhas been reported (Patent Document 3, Patent Document 4, and Non-PatentDocument 7). In this technique, plural point mutations responsible forlong-term persistence are identified in genome of the virus which is amaterial of the vector, and it is indicated that these mutations areinvolved in avoidance of activation of the innate immune system(deterioration in interferon expression). Also by deleting three genesfrom virus genome, and carrying new genes, it is possible to expressfour exogenous genes simultaneously. Further, it has been reported thatvectors are actively removed from cells by suppressing prey of L genethat encodes an RNA-dependent RNA polymerase by short interfering RNA(siRNA). It has been reported that the size of gene that can beexpressed with the use of a vector based on a mutant Sendai viruscapable of persistent infection is 4774 base pairs (sum of four genes,KLF4, OCT4, SOX2, and c-MYC) (Patent Document 3, Patent Document 4, andNon-Patent Document 7).

7. Future Challenge in Plural Gene Introducing Techniques

In existent gene introduction/expression techniques using RNA as aplatform for gene expression as described in the above section 6-4.,cell-reprogramming using four to five genes has been reported. Amongthese techniques, a defective and persistent expression Sendai virusvector described in the above section 6-4-2. has the most excellentcharacteristic, however, the number of genes that can be installed onthe vector has been reported to be at most four. In the technique usingan RNA virus as a material, it is difficult to alter the level of geneexpression.

As shown in the above section 6., the technique of externallyintroducing plural genes into animal cells including human cells andpersistently expressing the genes in the cells has been variouslymodified toward optimization for cell-reprogramming that converts thecharacteristics of normal tissue cells using genes, and produces usefulcells. However, a technique satisfying all the five requirementsrequired for ideal reprogramming shown in the above section 5. has notbeen reported heretofore.

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SUMMARY OF THE INVENTION Problems to be Solved by the Invention

As described above, a problem to be solved by the present invention isdeveloping a gene introduction/expression technique desired forreprogramming cells including human cells by the use of genes, and avector for the technique. It is also an object of the present inventionto provide a vector capable of carrying a total length of 5,000 or morenucleotides or at least six or more exogenous genes besidesreprogramming genes, and capable of persistently expressing the geneswithout activating an innate immune system in animal cells. Alsoprovided is an efficient technique for carrying six or more exogenousgenes on a vector.

Also provided is a gene introduction/expression technique satisfying therequirements (1) to (5) that are desired especially for reprogrammingtechnology, preferably satisfying the requirements further including therequirements (6) and (7).

(1) Capability of efficiently introducing exogenous genes into animalcells including human peripheral blood cells.

(2) Capability of persistently expressing the genes for any requiredperiod.

(3) Capability of avoiding the innate immune system possessed by cellsin expression of the genes.

(4) Capability of expressing the genes even if the total length of theintroduced exogenous genes is 5,000 or more nucleotides, desirably 8,000or more nucleotides.

(5) Capability of simultaneously expressing at least six, desirablyeight or more genes.

(6) Capability of regulating the levels of the expression of the genes.In particular, when plural genes are introduced, the expression levelcan be regulated individually.

In applying, in particular, to transplantation techniques, the followingpoint is also important.

(7) Capability of removing the gene expression system by a simpletechnique when the genes are no longer necessary.

Means for Solving the Problems

As described in the sections 6-1. to 6-3. of the background art, whenDNA is used as a platform for gene expression, it is theoretically verydifficult to satisfy all the five requirements, more preferably all theseven requirements required for ideal reprogramming shown as the“Problems to be Solved by the Invention”. On the other hand, asdescribed in the section 6-4., when RNA is used as a platform for geneexpression, it becomes the primary issue how to avoid the problem ofactivation of the intracellular innate immune system caused by avirus-derived RNA while increasing the number of genes that can beinstalled on the single vector or more, and the total length of genes to5,000 or more nucleotides.

Thus, the present invention, first, using mRNA fragments derived fromanimal cells that does not activate an innate immune system asmaterials, a negative-sense single-stranded RNA in which the RNAfragments are combined with transcription start signals, transcriptiontermination signals, and a replication origin that are recognized by anRNA-dependent RNA polymerase was designed. Then in the negative-sensesingle-stranded RNA, genes encoding four proteins required fortranscription and replication such as an RNA-dependent RNA polymerasewere installed after the structures thereof were optimized so as not tobe recognized as foreign substances by the innate immune system.Further, the present inventors developed a novel method of binding tengenes as designed by using five restriction endonucleases, and ten cRNAscomplementary to these ten genes were bound and then installed on thenegative-sense single-stranded RNA.

The present inventors succeeded in carrying at least ten exogenous genes(a total size of at least 13.5 kilo nucleotides) and expressing thempersistently for a long term without activating the innate immune systemby using the negative-sense single-stranded RNA completed by the abovemethod as a platform for gene expression. Further, the present inventorsmade the levels of expression of the installed genes regulatable withinthe range of up to 80 times by modifying the expression efficiency of Nprotein or C protein required for gene expression. Thus, by eliminatingthe RNA elements having a structure derived from virus as much aspossible, the present inventors succeeded in preparing a novel geneexpression system greatly beyond the limit of the capability of theconventional gene expression system using genome of RNA virus.

Further, by expressing an envelope protein and a matrix protein ofparamyxovirus in cells transfected with the negative-sensesingle-stranded RNA carrying exogenous genes, prepared in the presentinvention, according to the method described in Patent Document 3, Non-Patent Document 33, and Non-Patent Document 7, a particle thatencapsulates the RNA molecule, and has activity of introducing the RNAmolecule into another cell was prepared. This particle couldpersistently express ten genes installed on the RNA molecule whilekeeping activation of the innate immune system low in various animalcells including human blood cells. Further, by introducing siRNA that iscomplementary to the gene of the RNA-dependent RNA polymerase installedon the RNA molecule and of which structure has been optimized, into thecells, the RNA molecule carrying exogenous genes could be eliminated. Inthe manner as described above, the present inventors confirmed that allthe seven requirements including the five requirements (1) to (5) shownin the “Problems to be Solved by the Invention” and the requirements (6)and (7) in the aforementioned preferable case could be satisfied, andaccomplished the present invention.

Since the RNA molecule used in the present invention lacks a specificstructure required for the innate immune system to recognize as“pathogen-associated molecular pattern, PAMP”, the RNA molecule isdifficult to be captured by the innate immune system, namely it is“stealthy”. Therefore, hereinafter, the RNA molecule is referred to as“stealthy RNA”, the gene expression system using the RNA as a materialis referred to as “stealth RNA gene expression system”, the constructincluding the gene expression system and having the activity ofintroducing the gene expression system into animal cells is referred toas “stealth RNA vector”.

In other words, the present invention can be described as follows:

[1] A stealth RNA gene expression system comprising:

a negative-sense single-stranded RNA (A) having RNA sequences (1) to (8)below,

a single-stranded RNA binding protein (B), and

an RNA-dependent RNA polymerase (C),

wherein the stealth RNA gene expression system is a complex that doesnot activate an innate immune system:

(1) target RNA sequences encoding any given protein or functional RNA,

(2) RNA sequences constituting noncoding region(s) and derived frommRNA(s) expressed in animal cells,

(3) transcription start signal sequences recognized by the RNA-dependentRNA polymerase,

(4) transcription termination signal sequences recognized by thepolymerase enzyme,

(5) RNA sequences containing replication origins recognized by thepolymerase enzyme,

(6) RNA sequences encoding the polymerase enzyme with codons optimizedfor a biological species from which cells for transfection are derived,

(7) an RNA sequence encoding a protein that regulates activity of thepolymerase enzyme with codons optimized for a biological species fromwhich cells for transfection are derived, and

(8) an RNA sequence encoding the single-stranded RNA binding proteinwith codons optimized for a biological species from which cells fortransfection are derived.

Here, since typical cells for transfection are human cells, thepreferred cases can be described as follows.

[1′] A stealth RNA gene expression system comprising:

a negative-sense single-stranded RNA (A) having RNA sequences (1) to (8)below,

a single-stranded RNA binding protein (B), and

an RNA-dependent RNA polymerase (C),

wherein the stealth RNA gene expression system is a complex that doesnot activate an innate immune system:

(1) target RNA sequences encoding any given protein or functional RNA,

(2) human mRNA-derived RNA sequences constituting noncoding region(s),

(3) transcription start signal sequences recognized by the RNA-dependentRNA polymerase,

(4) transcription termination signal sequences recognized by thepolymerase enzyme,

(5) RNA sequences containing replication origins recognized by thepolymerase enzyme,

(6) RNA sequences encoding the polymerase enzyme with codons optimizedfor human cells,

(7) an RNA sequence encoding a protein that regulates activity of thepolymerase enzyme with codons optimized for human cells, and

(8) an RNA sequence encoding the single-stranded RNA binding proteinwith codons optimized for human cells.

[2] The stealth RNA gene expression system according to the [1], whereinthe target RNA sequences of the (1) contain at least six genes, or areRNA sequences having a total length of 5000 or more nucleotides.

Here, the target RNA sequences can contain seven to ten genes, or areRNA sequences having a total length of 5,000 to 15, 000 nucleotides.

[3] The stealth RNA gene expression system according to the [1] or [2],wherein the RNA sequences of the (2) are derived from mRNA of humangene(s) and each of the RNA sequences is having a length of 5 to 49nucleotides.

Here, as the mRNA sequence of a human gene, preferably a mRNA sequenceof a human House-keeping gene, more preferably a noncoding regionsequence in a mRNA sequence of a human House-keeping gene, for example,an RNA sequence described in (Table 1), or a partial sequence having alength of consecutive 5 to 49 nucleotides thereof, or a plurality ofthese sequences linked to each other can be used.

[4] The stealth RNA gene expression system according to any one of the[1] to [3], wherein each of the RNA sequences of the (2) havingsequences identical to or different from one another is placed adjacentto 3′ terminal site and/or 5′ terminal site of each of gene sequencescontained in the target RNA sequences of the (1).

[5] The stealth RNA gene expression system according to any one of the[1] to [4], wherein.

the RNA-dependent RNA polymerase encoded by the RNA sequences of the (6)consists L protein and P protein derived from an RNA virus belonging toa paramyxovirus family,

the protein that regulates activity of the polymerase enzyme encoded bythe RNA sequence of the (7) is C protein derived from the same virus asthe RNA virus,

the single-stranded RNA binding protein encoded by the RNA sequence ofthe (8) is NP protein derived from the same virus as the RNA virus, and

all of the RNA sequences of the (3) to (5) are RNA sequences containinga transcription start signal, a transcription termination signal, or areplication origin sequence derived from a genome of the same virus asthe RNA virus.

[6] wherein the RNA sequences encoding the L protein, P protein, Cprotein and NP protein are optimized for human cells, and have a GCcontent adjusted within a range of 50 to 60%.

[7] The stealth RNA gene expression system according to the [6], whereinthe RNA virus belonging to a paramyxovirus family is an RNA virusselected from the group consisting of Sendai virus, human parainfluenzavirus, and Newcastle disease virus.

[8] The stealth RNA gene expression system according to any one of the[1] to [7], wherein the transcription start signal sequences of the (3)are RNA sequences selected from the group of RNA sequences consisting of3′-UCCCACUUUC-5′ (SEQ ID NO: 1), 3′-UCCCUAUUUC-5′ (SEQ ID NO: 2),3′-UCCCACUUAC-5′ (SEQ ID NO: 3), 3′-UCCUAAUUUC-5′ (SEQ ID NO: 7), and3′-UGCCCAUCUUC-5′ (SEQ ID NO. 9), and the transcription terminationsignal sequences of the (4) are RNA sequences selected from the group ofRNA sequences consisting of 3′-AAUUCUUUUU-5′ (SEQ ID NO: 4),3′-CAUUCUUUU-5′ (SEQ ID NO: 5), 3′-UAUUCUUUUU-5′ (SEQ ID NO: 6), and3′-UUAUUCUUUUU-5′ (SEQ ID NO: 8).

[9] The stealth RNA gene expression system according to any one of the[4] to [8], wherein each of the transcription start signal sequences ofthe (3) having sequences identical to or different from one another isplaced adjacent to 3′ terminal site of each of the RNA sequence of the(2) that is placed adjacent to 3′ terminal site of each of genesequences contained in the target RNA sequences of the (1), and each ofthe transcription termination signal sequences of the (4) is placedadjacent to 5′ terminal site of the RNA sequence that is placed adjacentto 5′ terminal site of each of gene sequences contained in the targetRNA sequences of the (1).

[10] The stealth RNA gene expression system according to any one of the[7] to [9], wherein the RNA sequences containing a replication originsof the (5) contain the following sequences:

(a) an RNA sequence represented by (SEQ ID NO: 11) 3′-UGGUCUGUUCUC-5′ or(SEQ ID NO: 12) 3′-UGGUUUGUUCUC-5′, (b) an RNA sequence represented by(SEQ ID NO: 13) 3′-GAGAACAGACCA-5′ or (SEQ ID NO: 14)3′-GAGAACAAACCA-5′, (c) an RNA sequence represented by (SEQ ID NO: 15)3′-(CNNNNN)₃-5′, and (d) an RNA sequence represented by (SEQ ID NO: 10)3′-(NNNNNG)₃-5′.

[11] The stealth RNA gene expression system according to the [10],wherein the RNA sequence of the (a) is positioned at the 3′ terminus ofthe negative-sense single-stranded RNA (A), and the RNA sequence of the(b) is positioned at the 5′ terminus.

[12] The stealth RNA gene expression system according to the [10] or[11], herein the RNA sequence of the (c) starts at 79th nucleotide fromthe 3′ terminus of the negative-sense single-stranded RNA (A), and theRNA sequence of the (d) starts at 96th nucleotide from the 5′ terminus.

[13] The stealth RNA gene expression stem according to any one of the[10] to [12], wherein the RNA sequence containing replication origins ofthe (5) further contain in a position of 97th to 116th nucleotides fromthe 3′ terminus of the negative-sense single-stranded RNA (A), an RNAsequence of (e) 3′-AAAGAAACGACGGUUUCA-5′ (SEQ ID NO: 17) or an RNAsequence having the same length of 18 nucleotides as the (e).

[14] A stealth RNA vector including a complex composed of the stealthRNA gene expression system according to any one of the [1] to [13], andhaving activity of introducing the complex into animal cells, that doesnot activate an innate immune system.

[15] The stealth RNA vector according to the [14], that forms a virusparticle having ability to infect animal cells.

[16] An animal cell transfected with the stealth RNA vector according tothe [14] or [15].

[17] A stealth RNA which is a negative-sense single-stranded RNA (A)having RNA sequences of (1) to (8) below, capable of forming a complexthat does not activate an innate immune system together with asingle-stranded RNA binding protein (B), and an RNA-dependent RNApolymerase (C):

(1) target RNA sequences encoding any given protein or functional RNA,

(2) RNA sequences constituting noncoding region(s) that isunrecognizable by an innate immune system,

(3) transcription start signal sequences recognized by an RNA-dependentRNA polymerase,

(4) transcription termination signal sequences recognized by thepolymerase enzyme,

(5) RNA sequences containing replication origins recognized by thepolymerase enzyme,

(6) RNA sequences encoding the polymerase enzyme and having a structureoptimized to be unrecognizable by an innate immune system,

(7) an RNA sequence encoding a protein that regulates activity of thepolymerase enzyme, and having a structure optimized to be unrecognizableby an innate immune system, and

(8) an RNA sequence encoding a single-stranded RNA binding protein andhaving a structure optimized to be unrecognizable by an innate immunesystem.

The present invention also includes the following modes.

[17′] A stealth RNA which is a negative-sense single-stranded RNA (A)having RNA sequences of (1) to (8) below, capable of forming a complexthat does not activate an innate immune system together with asingle-stranded RNA binding protein (B), and an RNA-dependent RNApolymerase (C):

(1) target RNA sequences encoding any given protein or functional RNA,

(2) RNA sequences constituting noncoding region(s) and derived frommRNA(s) expressed in animal cells,

(3) a transcription start signal sequence recognized by theRNA-dependent RNA polymerase,

(4) transcription termination signal sequences recognized by thepolymerase enzyme,

(5) RNA sequences containing replication origins recognized by thepolymerase enzyme,

(6) an RNA sequence encoding the polymerase enzyme with codons optimizedfor a biological species from which cells for transfection are derived,

(7) an RNA sequence encoding a protein that regulate activity of thepolymerase enzyme with codons optimized for a biological species fromwhich cells for transfection are derived, and

(8) an RNA sequence encoding the single-stranded RNA binding proteinwith codons optimized for a biological species from which cells fortransfection are derived.

[17″] A stealth RNA which is a negative-sense single-stranded RNA (A)having RNA sequences of (1) to (8) below, capable of forming a complexthat does not activate an innate immune system together with asingle-stranded RNA binding protein (B), and an RNA-dependent RNApolymerase (C):

(1) target RNA sequences encoding any given protein or functional RNA,

(2) human mRNA-derived RNA sequences constituting noncoding region(s),

(3) transcription start signal sequences recognized by the RNA-dependentRNA polymerase,

(4) transcription termination signal sequences recognized by thepolymerase enzyme,

(5) RNA sequences containing replication origins recognized by thepolymerase enzyme,

(6) RNA sequences encoding the polymerase enzyme with codons optimizedfor human cells,

(7) RNA sequence encoding a protein that regulates activity of thepolymerase enzyme with codons optimized for human cells, and

(8) an RNA sequence encoding the single-stranded RNA binding proteinwith codons optimized for human cells.

[18] The stealth RNA according to the [17], wherein RNA sequencescontaining replication origins recognized by the RNA-dependent RNApolymerase of the located at the 3′ terminal site and the 5′ terminalsite of the negative-sense single-stranded RNA (A), and the RNA sequencelocated at the 3′ terminal site and the RNA sequence located at the 5′terminal site include RNA sequences complementary to each other.

[19] The stealth RNA according to the [17] or [18], wherein each of thetranscription start signal sequences of the (3) having sequencesidentical to or different from one another is placed adjacent to 3′terminal site of each of the RNA sequences of the (2) that is placedadjacent to 3′ terminal site of each of plural gene sequences containedin the target RNA sequences of the (1), and each of the transcriptiontermination signal sequences of the (4) is placed adjacent to 5′terminal site of the RNA sequence that is placed adjacent to 5′ terminalsite of each of plural gene sequences contained in the target RNAsequences of the (1).

[20] The stealth RNA according to any one of the [17] to [19], whereineach of the transcription start signal sequences of the (3) havingsequences identical to or different from on another is placed adjacentto 3′ terminal site of each of the RNA sequences of the (2) that isplaced adjacent to 3′ terminal site of each of plural gene sequencescontained in the target RNA sequences of the (1); each of thetranscription termination signal sequences of (4) is placed adjacent to5′ terminal site of the RNA sequence that is placed adjacent to 5′terminal site of each of plural gene sequences contained in the targetRNA sequences of the (1); and both of them constitute a cassettestructure together with restriction sites located at both ends of thecassette that can be cleaved by plural restriction endonucleases, andplural cassette structures are bound to each other.

[21] A method for reconstituting a stealth RNA gene expression system,comprising the following processes (1) to (5):

(1) preparing an Escherichia coli expressing T7 RNA polymerase;

(2) introducing into the Escherichia coli host of the (1), at least avector for Escherichia coli carrying an RNA encoding an RNA-dependentRNA polymerase and an DNA binding protein, and a vector for Escherichiacoli for expressing a DNA encoding RNA binding protein, together withthe negative-sense single-stranded RNA (A) according to any one of the[1] to [13] to transform the host,

(3) forming a complex of the negative-sense single-stranded RNAcontaining exogenous gene RNA expressed by T7 RNA polymerase, and RNAbinding protein in the transformed Escherichia coli of the (2),

(4) preparing animal cells in which an RNA-dependent RNA polymerase isexpressed, and

(5) introducing the complex of the negative-sense single-stranded RNAand the RNA binding protein obtained in the (3) into an animal cell hostof the (4) to reconstitute a stealth RNA gene expression system composedof the negative-sense single-stranded DNA, and the complex of the RNAbinding protein and the RNA-dependent RNA polymerase.

[22] A DNA-based tandem cassette having two cloning sites A and B,

the tandem cassette being composed of (1) multimerization site A, (2)transcription start signal A, (3) noncoding sequence A1, (4) cloningsite A, (5) noncoding region A2, (6) transcription termination signal A,(7) transcription start signal B, (8) noncoding sequence B1, (9) cloningsite B, (10) noncoding region B2, (11) transcription termination signalB, and (12) multimerization site B in order from the 5′ terminus,

the multimerization site A of the (1), and multimerization site B of the812) being DNAs that are identical to or different from each other andeach containing a recognition site by restriction endonuclease and or arecognition site by site-specific recombinase,

the transcription start signal A of the (2), and transcription startsignal B of the (7) being DNAs that are identical to or different fromeach other and each containing a transcription start signal recognizedby the RNA-dependent RNA polymerase when transcribed to RNA,

the noncoding sequence A1 of the (3), noncoding region A2 of (5),noncoding sequence B1 of the (8), and noncoding region B2 of the (10)being DNAs that are identical to or different from one another and eachbecoming RNA that is not recognized by an innate immune system of a hostcell when transcribed to RNA,

the cloning site A of the (4), and cloning site B of the (9) being DNAsthat are identical to or different from each other and each containingone or more recognition site by restriction endonuclease and/orrecognition site by site-specific recombinase,

the transcription termination signal A of the (6), and transcriptiontermination signal B of the (11) being DNAs that are identical to ordifferent from each other and each containing a transcriptiontermination signal recognized by the RNA-dependent RNA polymerase whentranscribed to RNA.

[23] The tandem cassette accord to the [22], wherein

the cloning site A of the (4) contains a recognition site by restrictionendonuclease A, and a recognition site by restriction endonuclease C inorder from 5′ terminal side, and

the cloning site B of the (9) contains a recognition site by restrictionendonuclease D, and a recognition site restriction endonuclease B inorder from 5′ terminal side,

provided that the restriction endonuclease A and the restrictionendonuclease D give single-stranded protruding ends of the samesequence, and the restriction endonuclease C and the restrictionendonuclease B give single-stranded protruding ends of the samesequence.

[24] The tandem cassette to according to the [22] or [23], wherein bothof the multimerization site A of the (1), and multimerization site B ofthe (12) are DNAs containing a recognition site a restrictionendonuclease giving a single-stranded protruding end of any sequencerepresented by NN or NNN.

[25] The tandem cassette according to any one of the [22] to [24],wherein the noncoding sequence A1 of the (3), noncoding region A2 of the(5), noncoding sequence B1 of the (8), and noncoding region B2 of the(10) are identical to or different from one another and each of them iscDNA corresponding to a partial sequence of RNA sequence derived frommRNA expressed in animal cells, and

one of human-derived genes identical to or different from each other isinserted into the cloning site A of the (4), and cloning site B of the(9).

Effects of the Invention

Since the stealth RNA gene expression system of the present invention isdifficult to be captured by the innate immune system, it has a very lowcytotoxicity, and is capable of carrying ten genes and introducing theminto various tissue cells, and expressing them persistently for anyrequired period. The wording of “capable of avoiding an innate immunesystem” or “not recognized by an immune system” used herein means thatthe introduced gene or the vector or the like used for introduction doesnot substantially stimulate the innate immunity of the host.Specifically, it means that the interferon β inducibility as an index is30 or less, preferably 20 or less, more preferably 10 or less, when theexpression amount of IFN-β mRNA in normal cells is 1.0.

Also, since the stealth RNA, gene expression system functions incytoplasm, by using the stealth RNA vector including the gene expressionsystem, it is possible to introduce and express the installed genes intocells of peripheral blood not having proliferating ability and have notundergone cell division. Furthermore, a gene expression system withvarious expression intensity within a maximum of 80 times can beselected, and easy removal is allowed by suppressing activity of theRNA-dependent RNA polymerase if no longer necessary. Therefore, thistechnique is suited for the object of efficiently reprogrammingcharacteristics of animal cells including human cells by using six ormore genes, that has been impossible heretofore.

For example, application of efficiently preparing iPS cells having highquality for clinical use in regenerative medicine under such a severecondition not containing animal derived components (Xeno-free) and notusing feeder cells (Feeder-free) using human peripheral blood cells as amaterial can be conceived. Also, application to the technology calleddirect reprogramming for creating useful cells such as nerve cells,neural stem cells, stem cells, pancreatic beta cells and the like fromhuman tissue cells (blood, skin, placenta, etc.) using six or more genesis enabled. Further, since the possibility of causing cell death orinflammation is low, application to gene therapy by various genesincluding giant genes, and application to regenerative medicine by invivo reprogramming are expected.

Since the stealth RNA gene expression system can carry plural genessimultaneously, and express them in a certain ratio, it is alsoeffective in production of biopharmaceuticals made up of pluralsubunits. For example, production of human immunoglobulin G, it isnecessary that each subunit is expressed simultaneously in the samecell. It is also required that H-chain and L-chain are expressedsimultaneously in a ratio 1:1 in the same cell in production of humanimmunoglobulin G, and H-chain, L-chain, and μ-chain are expressedsimultaneously in a ratio of 1:1:0.2 in the same cell in production ofhuman immunoglobulin M. The stealth RNA gene expression system caneasily satisfy such a requirement.

Further, since the level of gene expression can be varied in the stealthRNA gene expression system, strong gene expression required forproduction of biopharmaceuticals can be easily realized. Conventionalmanufacturing process of biopharmaceuticals using animal cells requiresthe process of establishing a stable cell strain in which the number ofcopies of the gene integrated into chromosome is amplified, whichrequires large amounts of time and labor. However, by employing thestealth RNA gene expression system, such labor is no longer required.

Also, the stealth RNA gene expression system is effective forsuppressing gene mutation which is problematic in production ofbiopharmaceuticals. Recently, it has been reported that the primarycause of occurrence of mutation genome of an RNA virus is cytosolicadenosine deaminase (Adenosine deaminase acting on RNA, ADAR1)(Non-Patent Document 39). Since ADAR1 is induced by activation of theinnate immune system, it is possible to suppress mutation of genes inthe stealth RNA gene expression system by controlling induction of ADAR1as low as possible.

The stealth RNA gene expression system is also suited for expression ofa drug-discovery target protein made up of plural subunits. For example,for expression of NADPH oxidase (Nox2) which is a drug-discovery targetenzyme, it is necessary to simultaneously express six subunits,gp91phox, p22phox, Rac, p47phox, p67phox, and p40phox, and this can beeasily realized by the stealth RNA gene expression system. Further, byusing the stealth RNA vector, it is possible to express thedrug-discovery target protein in target cells such as primary culturevascular endothelial cells and nerve cells for which gene introductionand expression has been difficult because they do not undergo celldivision, and it is possible to achieve the object easily.

Further, since the stealth RNA gene expression system and the stealthRNA vector are less likely to cause cell injury or inflammation, theycan be applied as a platform of gene therapy for obtaining a therapeuticeffect by in vivo gene expression. In particular, since the stealth RNAgene expression system and the stealth RNA vector can carry andpersistently express a giant gene such as cDNA of blood coagulationfactor VIII which is a product of a gene responsible for hemophilia A(7053 nucleotides) and cDNA of dystrophin which is a product of a generesponsible for Duchenne muscular dystrophy (11058 nucleotides) unlikeconventional gene introduction/expression vectors, application asvectors for gene therapy of these diseases is expected.

Further, since the tandem cassette used in a tandem cassette linkingmethod developed for carrying six or more, preferably eight or moreexogenous genes on the vector of the present invention is constructed ona DNA basis, the present technique can be widely applied to common DNAexpression vectors besides the stealth RNA vector of the presentinvention.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 illustrates a structure of a negative-sense single-stranded RNAmolecule prepared by combining RNA derived from mRNA expressed in animalcells, and transcription start signals, transcription terminationsignals, and replication origins recognized by an RNA-dependent RNApolymerase.

FIG. 2 illustrates structures of 3′ terminus and 5′ terminus of anucleic acid required for replication of a negative-sensesingle-stranded RNA molecule.

FIG. 3 illustrates a structure of 3′ terminus of a nucleic acid requiredfor replication of a negative-sense single-stranded RNA molecule.

FIG. 4 illustrates a structure of 5′ terminus of nucleic acid requiredfor replication of a negative-sense single-stranded RNA molecule.

FIG. 5 illustrates analysis of codon adaptation index in mRNAs derivedfrom RNA viruses.

FIG. 6 illustrates analysis of GC contents in mRNAs derived from RNAviruses.

FIG. 7 illustrates a method of designing an exogenous gene cDNA to beinstalled on a stealth RNA gene expression system.

FIG. 8 illustrates a method of connecting two exogenous gene cDNAs.

FIG. 9 illustrates a method of connecting ten exogenous gene cDNAs.

FIG. 10 illustrates a method of constructing a template cDNA forpreparing a stealth RNA gene expression system into which ten exogenousgenes are incorporated.

FIG. 11 illustrates a first method for reconstituting a stealth RNA geneexpression system from a template cDNA.

FIG. 12 illustrates a second method for reconstituting a stealth RNAgene expression system from a template cDNA.

FIG. 13 illustrates a genome structure of a stealth RNA gene expressionsystem carrying ten exogenous gene cDNAs.

FIG. 14 illustrates gene expression activity of a stealth RNA geneexpression system carrying ten exogenous gene cDNAs.

FIG. 15 illustrates a genome structure of a stealth RNA gene expressionsystem carrying ten exogenous gene cDNAs, prepared while the basesequences of the nucleic acid are optimized in a different manner fromFIG. 13.

FIG. 16 illustrates a genome structure of a stealth RNA gene expressionsystem carrying ten exogenous gene cDNAs, prepared while the arrangementof N, C, PolS (P) genes is changed.

FIG. 17 illustrates interferon induction activity of a stealth RNAvector.

FIG. 18 illustrates a genome structure of a stealth RNA gene expressionsystem carrying an additional factor for completely avoiding an innateimmunity inducibility.

FIG. 19 illustrates interferon inducibility by a stealth RNA vectorcarrying an additional factor.

FIG. 20 illustrates structures of stealth RNA gene expression systemshaving different gene expression levels (indicated by positive-sense RNAsequences).

FIG. 21 illustrates a genome structure and gene expression of a stealthRNA gene expression system in which C gene is deleted or translation ofC gene is suppressed.

FIG. 22 illustrates activity of packaging signal of a stealth RNA geneexpression system.

FIG. 23 illustrates removal of a stealth RNA gene expression system fromcells.

FIG. 24 illustrates genome structures of stealth RNA vectors preparedheretofore.

FIG. 25 illustrates a preparation efficiency of induced pluripotent stemcells (iPS cells) by a stealth RNA vector carrying six reprogramminggenes.

FIG. 26 illustrates expression of immunoglobulin M by a stealth RNA geneexpression system.

FIG. 27 illustrates expression of a bi-specific antibody molecule by astealth RNA gene expression System.

DESCRIPTION OF EMBODIMENTS 1. Constituents of “Stealth RNA GeneExpression System” of the Present Invention

The RNA molecule used in the present invention is “stealthy”, namely itis difficult to be captured by the innate immune system. Therefore, inthe present invention, the RNA molecule is referred to as “stealthyRNA”, the gene expression system using the RNA as a material is referredto as “stealth RNA gene expression system”, the structure including thegene expression system and having the activity of introducing the geneexpression system into animal cells is referred to as “stealth RNAvector”.

A stealth RNA gene expression system in the present invention is acomplex that includes a negative-sense single-stranded RNA (A)containing RNA sequences (1) to (8) below, a single-stranded RNA bindingprotein (B), and an RNA-dependent RNA polymerase (C), and not activatean innate immune system. A stealth RNA vector is a particle thatcontains the complex and has activity of introducing the complex intoanimal cells. In the present invention, a sequence encoding proteinmeans an RNA sequence of the anti sense strand in describing an RNAsequence of negative-sense single-stranded RNA.

(1) target RNA sequences encoding any given protein or functional RNA,

(2) RNA sequences constituting noncoding region(s) that isunrecognizable an innate immune system,

(3) a transcription start signal sequences recognized by theRNA-dependent RNA polymerase,

(4) transcription termination signal sequences recognized by thepolymerase enzyme,

(5) RNA sequences containing replication origins recognized by thepolymerase enzyme,

(6) RNA sequences encoding the polymerase enzyme and having a structureoptimized to be unrecognizable by an innate immune system,

(7) an RNA sequence encoding a protein that regulates activity of thepolymerase enzyme and having a structure optimized to be unrecognizableby an innate immune system, and

(8) an RNA sequence encoding the single-stranded RNA binding protein andhaving a structure optimized to be unrecognizable by an innate immunesystem.

(Hereinafter, also referred to as gene RNA or simply referred to asgene.)

Here, each of the RNA sequences of (2) preferably has a length of 5 to49 nucleotides, and is placed as a noncoding region on 3′ terminal sideand 5′ terminal side of each of the introduced exogenous gene RNAs (1).

While the stealth RNA gene expression system functions even when theintroduced exogenous gene RNA of (1) contains less than six genes, forexample, one to five genes, or contains a total nucleotide length ofless than 5,000 nucleotides, the RNA gene expression system of thepresent invention exerts a significant effect in particular, when theexogenous gene RNA contains six or more, preferably eight or more, morepreferably ten or more genes, or contains RNA of a total nucleotidelength of 5,000 nucleotides, preferably 8,000 nucleotides, and morepreferably 10,000 nucleotides.

In this description, the wording “gene or gene material” includes anegative-sense RNA or cDNA, and a positive-sense RNA or cDNA that iscomplementary to the same. In other words, those capable of synthesizingany of the gene or gene material by transcription or reversetranscription are also included in the present invention.

2. Constituents of Stealth RNA Expression System of the PresentInvention 2-1. Preparation of Tandem Cassette for Introduction ofExogenous Gene RNA

The exogenous gene RNA in the stealth RNA gene expression system of thepresent invention have “(2) RNA sequences that are not recognized by aninnate immune system” within 3′ terminal and 5′ terminal noncodingregions thereof, wherein each of the RNA sequences is identical to ordifferent from each other and having a length of 5 to 49 nucleotide, andcan be prepared as a cassette by providing a “transcription startsignal” of (3) and a “transcription termination signal” of (4) onfurther outer 3′ terminal and 5′ terminal site respectively, andproviding multimerization sites at both outermost terminals.

The negative-sense single-stranded RNA used in the stealth RNA geneexpression system of the present invention can be easily constructed byusing the DNA-based tandem cassette shown below.

The tandem cassette of the present invention is composed of (1)multimerization site A, (2) transcription start signal A, (3) noncodingsequence A1, (4) cloning site A, (5) noncoding region A2, (6)transcription termination signal A, (7) transcription start signal B,(8) noncoding sequence B1, (9) cloning site B, (10) noncoding region B2,(11) transcription termination signal B, and (12) multimerization site Bin order from the 5′ terminus. The tandem cassette is schematicallyshown in the lower diagram of FIG. 7.

The multimerization sites A and B may be identical to or different fromeach other, and any sequence can be used as long as it can be used formultimerization of the cassette or for binding with other nucleic acid.Preferred examples of the multimerization site include a restrictionsite by a restriction endonuclease, and a recognition site by asite-specific recombinase. Examples of preferred restrictionendonucleases include SapI, BbsI, BbvI, BcoDI, BfuAI, BsaI, BsmBI,BsmFI, BtgZI, EarI, FokI, HgaI, and SfaNI having a characteristic ofgenerating a single-stranded protruding end structure having anysequence indicated, for example, by NN or NNN on the terminus generatedby digestion. As other preferred examples, AlwNI, BglI, BstAPI, BstXI,DraIII, SfiI and so on having an indefinite sequence within therecognition site are recited. When homologous recombination is utilized,sequences such as attB1 and attB2 can be recited as a recognition siteby a recombinase. Further, when Gibson Assembly System (New EnglandBiolabs, Inc) is utilized, any sequence of 15 or more nucleotides can beused as a multimerization site providing that it has the same sequenceas the overlapping sequence at an end of other tandem cassette which isto be a counter part of linkage.

The transcription start signals A and B can be identical to or differentfrom each other, and can be any sequence as long as they are functionalas transcription start signals recognized by an RNA-dependent RNApolymerase when they are transcribed to RNA. Examples of thetranscription start signals recognized by an RNA-dependent RNApolymerase will be specifically described in the following paragraphs.Preferably, the sequences represented SEQ ID NOs: 1 to 3 can be recited.

The noncoding sequences A1, A2, B1 and B2 can be identical to differentfrom one another. Any sequence is acceptable as long as the sequencegives “RNA that is not recognized by innate immune system” defined inthe above when transcribed to RNA, and as a preferred example, sequenceshaving a length of 5 to 49 nucleotides and shown in Table 1 can berecited.

The cloning sites A and B can be any sequence that allows insertion of adesired exogenous gene. Preferably, one cloning site contains one or twoor more recognition sites by restriction endonucleases, or contains oneor two or more recognition sites by site-specific recombinases.Preferred examples of the cloning site include a sequence containingAcc65I recognition site and SalI recognition site, a sequence containingAcc65I recognition site and XhoI recognition site, a sequence containingBsiWI recognition site and SalI recognition site, and a sequencecontaining BsiWI recognition site and XhoI recognition site.

The transcription terminal signals A and B can be identical to ordifferent from each other, and can be any sequences as long as they arefunctional as transcription termination signals recognized by anRNA-dependent RNA polymerase when they are transcribed into RNA.Examples of the transcription termination signals recognized by anRNA-dependent RNA polymerase will be specifically described in thefollowing paragraphs. Preferably, the sequences represented by SEQ IDNOs: 4 to 6 can be recited.

In the one tandem cassette, at least two exogenous genes can beinserted. A cassette multimer formed of five linked cassettes eachhaving insertion of two exogenous genes carries ten exogenous genes. Inthe following Examples, DNA fragments carrying four, six, or tenexogenous genes are prepared by utilizing multimerization of tandemcassettes as described above and sub-cloned into plasmids. Further, bycombining with RNA-dependent RNA polymerase genes derived from virus,and an RNA binding protein gene, an RNA, expression system desired inthe present invention is constructed.

2-2. RNA-Dependent RNA Polymerase, and Transcription Start Signal andTranscription Termination Signal Recognized by the Polymerase Enzyme

Preferably, the “RNA-dependent RNA polymerase”, and the transcriptionstart signal and the transcription termination signal recognized by thepolymerase enzyme are selected from sequences derived from the samenegative-sense RNA virus, and are typically sequences derived fromgenome of a virus belonging to the paramyxovirus family. Since thecombination of “RNA-dependent RNA polymerase” of genome of a virusbelonging to the paramyxovirus family, “transcription start signalrecognized by the polymerase enzyme”, and “transcription terminationsignal recognized by the polymerase enzyme” has the same basicstructure, combinations of sequences derived from any virus can be used.

In Examples of the present invention, a combination of L protein (largesubunit of RNA polymerase, PolL) and P protein (small subunit of RNApolymerase, PolS) derived from Sendai virus was selected as“RNA-dependent RNA polymerase”, “3′-UCCCACUUUC-5′ (SEQ ID NO: 1)” wasselected as “RNA which is a transcription start signal recognized by anRNA-dependent RNA polymerase”, “3′-AAUUCUUUUU-5′ (SEQ ID NO: 4)” wasselected as “RNA which is a transcription termination signal recognizedby an RNA-dependent RNA polymerase”, and the transcription start signaland the transcription termination signal were placed respectively on 3′side and 5′ side of each gene (FIG. 1). Then C protein of Sendai virus(C) is used as “protein that regulates activity of RNA polymerase”, andNP protein of Sendai virus (N) is used as “single-stranded RNA bindingprotein”.

In the technique disclosed in the present description, since RNA is usedmainly in the form of a negative-sense single-stranded RNA, an RNAsequence is disclosed from terminal side as sequence information of anegative strand unless otherwise noted. However, sequence information inthe sequencing listings that forms part of the Present description isdescribed from 5′ terminal side according to the guidelines.

When a combination of L protein and P protein of Sendai virus isselected as “RNA-dependent RNA polymerase”, as a transcription startsignal, besides “3′-UCCCACUUUC-5′ (SEQ ID NO: 1)”, “3′-UCCCUAUUUC-5′(SEQ ID NO: 2)”, and “3′-UCCCACUUAC-5′ (SEQ ID NO: 3)”, RNA having anequivalent function as these sequences can be used. Similarly, also as atranscription termination signal, besides “3′-AAUUCUUUUU-5′ (SEQ ID NO:4)” “3′-CAUUCUUUUU-5′ (SEQ ID NO: 5)”, and “3′-UAUUCUUUUU-5′ (SEQ ID NO:6)”, RNA having an equivalent function as these sequences can be used.When a combination of L protein and P protein of human parainfluenzaviruses type 3 is selected as “RNA-dependent RNA polymerase”,“3′-UCCUAAUUUC-5′ (SEQ ID NO: 7)” or an RNA having an equivalentfunction can be used as a transcription start signal, and“3′-UUAUUCUUUUU-5′ (SEQ ID NO: 8)” or an RNA having an equivalentfunction can be used as a transcription termination signal. Further,when a combination of L protein and P protein of Newcastle disease virusis selectee as “RNA-dependent RNA polymerase”, “3′-UGCCCAUCUUC-5′ (SEQID NO: 9)” or an RNA having an equivalent function can be used as atranscription start signal, and “3′-AAUCUUUUUU-5′ (SEQ ID NO: 10)” anRNA having an equivalent function can be used as a transcriptiontermination signal.

2-3. Elements for Replication Function of Stealth RNA Gene ExpressionSystem of the Present Invention

Essential elements for the replication function of the stealth RNA geneexpression system of the present invention include replication originsrecognized by an RNA-dependent RNA polymerase, and sequences having thestructure of (CNNNNN)₃- on 3′ terminal site, and (NNNNNG)₃- on 5′terminal site.

In Examples of the present invention, since a combination of L proteinand P protein of Sendai virus was selected as “RNA-dependent RNApolymerase”, RNA of 114 nucleotides existing at 3′ terminus of thegenome of Sendai virus, and RNA of 96 nucleotides existing at 5′terminus of the genome of Sendai virus were selected as “RNA containinga replication origin recognized by an RNA-dependent RNA polymerase”.Among these structures, those essential for the replication function ofthe stealth RNA gene expression system are as follows (FIG. 2, FIG. 3,FIG. 4).

(1) “3′-UGGUCUGUUCUC-5′ (SEQ ID NO: 11)” existing at 3′ terminus of thegenome or an RNA sequence of 12 nucleotides having an equivalentfunction (for example, “3′-UGGUUUGUUCUC-5′ (SEQ ID NO: 12)”).

(2) “3′-GAGAACAGACCA-5′ (SEQ ID NO: 13)” existing at 5′ terminus of thegenome or an RNA sequence of 12 nucleotides having an equivalentfunction (for example, “3′-GAGAACAAACCA-5′ (SEQ ID NO: 14)”),

(3) an RNA sequence of 18 nucleotides having a structure of“3′-(CNNNNN)₃-5′ (SEQ ID NO: 15)” starting from the 79th nucleotide from3′ terminus of the genome, and

(4) an RNA sequence of 18 nucleotides having a structure of“3′-(NNNNNG)₃-5′ (SEQ ID NO: 15)” starting from the 96th nucleotide from5′ terminus of the genome.

Among these, (1) and (2) are considered as replication originsrecognized by an RNA-dependent RNA polymerase because they are mutuallycomplementary sequences, end then 3′ terminus of the genome RNA and 3′terminus of the antigenome RNA (RNA complementary to the genome RNA) areidentical. While the functions of (3) and (4) are unknown, it is knownthat they are the sequences essential for replications single-strandedRNA by an RNA-dependent RNA polymerase (Non-Patent Document 42).

2-4. Packaging Signal Region Essential for Particulation inNegative-Sense Single-Stranded RNA

In the present invention, the present inventors first identified theregion spanning from the 97th nucleotide to the 114th nucleotide of 3′terminus of the genome as a region that is a packaging signal forparticle formation in the negative -sense single-stranded RNA.

As shown in Example 18 (FIG. 22), when the whole or the region(indicated as “sequence D”) was deleted, the efficiency of particleformation of the stealth RNA vector was significantly deterioratedalthough gene expression in the packaging cell was not influenced.

This indicates that this sequence of 18 nucleotides, or the regionhaving a length of 18 nucleotides or a part thereof is a sequence orregion that is essential for incorporation into a virus-like particle.

Then the present inventors replaced this sequence of 18 nucleotides witha partial sequence that is arbitrarily selected from partial sequencesof mRNA derived from House-keeping gene recited in (Table 1) ((5) ofFIG. 3, SEQ ID NO: 75), and confirmed that the efficiency of particleformation was not changed.

On the basis of this result, it is considered that the region having alength of 18 nucleotides from the 97th to 114th nucleotides from 3′terminus of the genome or a region having a partial length thereof isessential for packaging for particle formation in the negative-sensesingle-stranded RNA. In other words, it can be concluded that the regionis “packaging signal region” that is not essential for transcription andreplication of the negative-sense single-stranded RNA as a template, butis essential for incorporation of the stealth RNA gene expression systeminto a virus-like particle.

(5) RNA having a length of 18 nucleotides, corresponding to“3′-AAAGAAACGACGGUUUCA-5′ (SEQ ID NO: 17)” from the 97th to 114thnucleotides from 3′ terminus of the genome, or any RNA having a lengthof at least consecutive 8 or more nucleotides, preferably 10 or morenucleotides, more preferably 15 more nucleotides thereof.

The possibility that the stealth RNA gene expression system lacking thelength of 18 nucleotides or a partial region thereof of the above (5)leads production of a virus-like particle containing the stealth RNAgene expression system is very low even if the host cells are infectedwith a homogeneous or heterogeneous virus.

Thus, the region having a length of 18 nucleotides or a partial regionthereof is an essential region when the stealth RNA gene expressionsystem of the present invention is prepared as an infectious particle,and used as a stealth RNA gene expression vector, however, the region iscontrarily a sequence that should be eliminated for biopharmaceuticalproduction where it is desired to ultimately eliminate contaminationwith virus-like particles and to ensure the safety.

2-5. Construction of Template for Gene Expression of Negative-SenseSingle-Stranded RNA

It is known that an RNA molecule carrying a combination of “DNA which isa transcription start signal recognized by an RNA-dependent RNApolymerase”, “DNA which is a transcription termination signal recognizedby an RNA-dependent RNA polymerase” and “RNAs containing a replicationorigin recognized by an RNA-dependent RNA polymerase” existing at 3′terminus and 5′ terminus of a negative-sense single-stranded RNA,together with any exogenous gene between the transcription start signaland the transcription termination signal serves as a template fortranscription or replication in the presence of essential factors suchas an RNA-dependent RNA polymerase derived from a virus supplied intrans (Non-Patent Document 43, Non-Patent Document 44, and Non PatentDocument 45). For example, it is demonstrated that a negative-sensesingle-stranded RNA having the aforementioned structure carrying acombination of a transcription start signal, a transcription terminationsignal and replication origins derived from Sendai virus, andChloramphenicol acetyltransferase (CAT) gene of Escherichia coli as aexogenous gene serves as a template for transcription and replication ina cell infected with Sendai virus to produce CAT (Non-Patent Document43, and Non-Patent Document 44). Also it is indicated that anegative-sense single-stranded RNA having an equivalent structure ispersistently replicated in cells in which NP (single-stranded RNAbinding protein) P (small subunit of RNA-dependent RNA polymerase) and L(large subunit of RNA-dependent RNA polymerase) proteins of Sendai virusare stably expressed. (Non-Patent Document 45).

These reports indicate that the negative-sense single-stranded RNAprepared in the present invention as a template for gene expression,however, when such a technique is used as it is, the activity oftranscription or replication depends on the NP, P, and L proteinssupplied in trans from the cells containing genes of the virus, so thata general gene expression system enabling gene expression in any cell isnot obtained. Thus, the present inventors attempted to carry genesrequired for transcription and replication on an RNA molecule having thestructure shown in the above section 2-3. (FIG. 1) and formed ofcomponents that are not recognized by an innate immune system.

3. Findings Regarding Avoidance of Activation of Innate Immune System(PAMP) in Animal Cells 3-1. Regarding PAMP of Virus-Derived RNA

An innate immune system possessed by an animal cell is activated byrecognition of a “molecular pattern characteristic of pathogenicmicroorganism (Pathogen-associated molecular pattern, PAMP)” existing ingenome RNA of a virus that has been entered inside the cell, or mRNA ofa virus gene. The structure of PAMP has been identified in hepatitis Cvirus and human immunodeficiency virus. In hepatitis C virus, it hasbeen reported that a uridine-rich sequence positioned in a noncodingregion at 3′ terminal of the genome is RAMP (Non-Patent Document 35).Meanwhile, in a human immunodeficiency virus, it has been reported thatthe region having a high adenine content existing in mRNA transcribedfrom three genes of Gag, Pol, and Env is PAMP (Non-Patent Document 36).Besides the above, in Sendai virus, strong PAMP activity is detected ina long-chain RNA fraction exceeding 600 nucleotides existing in infectedcells (Non-Patent Document 37), and the existence of a high secondarystructure that potentially functions as PAMP is known also in noncodingregion of each gene of F, HN and L (Non-Patent Document 38). Thus, it isexpected that most of virus-derived RNAs contain PAMP.

3-2. Investigation of Optimization of Virus-Derived RNA

The attempt to disrupt the PAMP structure by optimizing codons of theregion encoding a protein in the RNA virus genome for human cells, andthus to avoid the activation of the innate immune system has been oftenconducted heretofore. For example, it has been reported that since PAMPexists in each mRNA transcribed from each of Gag, Pol, and Env genes ofhuman immunodeficiency virus (HIV), each gene induces interferon when itis expressed as it is in animal cells, whereas interferon induction issuppressed in each of Gag, Pol, and Env proteins that are optimized forhuman cells and expressed (Non-Patent Document 36). Also in a simianimmunodeficiency virus (SIV), likewise in HIV, it is known that PAMPexists in each mRNA of Gag, Pol, and Env, and by optimizing codons inthe region containing PAMP in each gene for human cells, the interferoninducibility decreases (Non-Patent Document 48). However, the interferoninducibility of SIV little changes only by optimization of codons in theregion containing PAMP in Pol gene in the SIV genome sequence. In lightof this, codons of the region containing PAMP of Gag gene were alsooptimized in addition to optimization of Pol gene, and this resulted inreduction in the replicability of the virus to 1% or less, andsignificant impairment in functions of transcription and replication ofthe virus (Non-Patent Document 48). This result not only reveals thatPol gene or Gag gene of SIV encodes Pol protein or Gag protein, but alsoreveals that the information required for the function of transcriptionor replication of the virus exists in the nucleic acid sequence itselfthat encodes the protein.

Also for the region containing PAMP in a noncoding region of 3′ terminusof the genome of hepatitis C virus, there is a report that the virusreplicability is impaired when the region is disrupted (Non-PatentDocument 82, and Non-Patent Document 83).

These results indicate that the “region containing PAMP” in RNA virusgenome is very likely to be also a region essential for the functionssuch as replication of the virus.

Thus, since a universal method for removing the structure having afunction of PAMP from genome nucleic acid without impairing the functionof the RNA virus is not known, application of the technique foroptimizing codons of the region containing PAMP in the virus RNA forhuman cells to an RNA virus vector contrarily leads a negative result.

3-3. Utilization of Virus Derived Innate Immunity Inhibitory Factor

In conventional techniques using genome of an RNA virus or a syntheticRNA as a platform for gene expression, the cytotoxicity is weakened byinhibiting activation of the innate immune system by PAMP by the actionof the factor competing the innate immune system possessed by variousviruses, rather than by elucidating the structure recognized as PAMP andremoving the structure. For example, B18R protein, which is used as anessential constituent in Non-Patent Document 26 and Non-Patent Document27, is an interferon binding protein encoded by genomic DNA of vacciniasvirus, and has a function of inhibiting activation of the innate immunesystem by inhibiting the activity of interferon.

Further, in the vectors based on Sendai virus described in PatentDocument 3, Patent Document 4, and Non-Patent Document 7, mutation of anRNA-dependent RNA polymerase (L protein and P protein), and expressionof V protein derived from Sendai virus serve to suppress the innateimmune system. V protein is one of proteins produced from mRNAtranscribed from P gene region of Sendai virus, and has an N-terminalregion (317 amino acid residues) common to that of P and a basicC-terminal region (67 amino acid residues) having a structure peculiarto V protein (Non-Patent Document 39). V protein inhibits activation ofthe innate immune system through inhibition of a transcription factorIRF-3 (Non-Patent Document 40). It is known that in a Vprotein-defective Sendai virus prepared by artificially introducingmutation into a base sequence of P gene, the function of suppressingactivation of the innate immune system is lost and the virus is easilyeliminated from the infected individual (Non-Patent Document 40, andNon-Patent Document 41).

In the case using a virus derived innate immunity inhibitory factortogether as described above, there arises a concern in safety that theinnate immune system cannot be activated even when the cells into whichthe exogenous gene is introduced are infected with other species ofpathogenic microorganism. For example, in cells stably retaining genomeof the Sendai virus vector, V protein is constantly expressed. Thus,when this vector is used in tissue cells of a living body, there is apossibility that the innate immune system cannot be activated even whenthe cells are infected with other virus. Therefore, a technique ofavoiding activation of an innate immune system by a method not relyingon suppression of the innate immune system by a virus derived factor isdesired.

4. Techniques for Avoiding RNA in RNA Gene Expression System of thePresent Invention 4-1. “RNA That is Not Recognized by an Innate ImmuneSystem” Found Within Noncoding Region Sequence

The key of the present invention is selection of RNA capable of avoidingactivation of an innate immune system possessed by animal cells. Asdescribed above, the wording “avoiding activation of an innate immunesystem” used in the present invention means that the interferon βinducibility as an index is 30 or less, preferably 20 or less, morepreferably 10 or less, when the expression amount of IFN-β mRNA innormal cells is 1.0.

Thus, in the present invention, as a material for “RNA that is notrecognized by an innate immune system”, the present inventors decided touse RNA, sequences derived from mRNA expressed in animal cells such ashuman cells, and selected mRNA derived from House-keeping genes that areexpressed in a wide variety of human cells. The mRNA is expressed inmost of human cells in relatively large quantity, and does not contain amotif recognized by the human innate immune system. Further, fromnoncoding regions in the mRNAs that not encore protein, RNAs each havinga length of 5 nucleotides to 49 nucleotides that do not form acomplicated secondary structure were selected (Table 1), and placed in anoncoding region on 5′ side aged in a noncoding region of 3′ side ofeach gene installed on the vector (FIG. 1).

All of the partial sequences of mRNA derived from House-keeping generecited below (Table 1) can be used as particularly preferred sequencesamong “RNA sequences derived from mRNA expressed in animal cells” or“RNA that is not recognized by an innate immune system” in the noncodingregion sequence of the present invention. As other examples of suchpreferred sequences, partial sequences of RNA sequences derived frommRNA of genes that are expressed in large quantity in a living body suchas albumin gene can also be preferably used.

As described above, in Examples of the present invention, noncodingregion sequences derived from mRNA expressed in human cells areselected, and partial sequences thereof were used in consideration ofapplication to regenerative medicine, however, “RNA that is notrecognized by an innate immune system” is not limited to the sequencesderived from noncoding region sequences derived from mRNA recited inExamples or (Table 1). For example, in OptimumGen Gene Design System(Patent Document 7, GenScript USA Inc.), partial sequences of anoncoding sequence possessed by human mRNA appropriately selected from agroup of human mRNAs that are highly expressed in human, employed fordetermining a standard CAI value can be used. Besides these, human RNAsother than mRNA, RNAs expressed in cells of other animal species, andnon-native synthetic RNAs can also be selected as long as they are notrecognized by the innate immune system of the host cells in which thevector is used.

TABLE 1 Animal species Position Position from which GenBank of insequence Name (abbreviated name) of gene from which Length accessionSequence cassette cassette is derived sequence is derived (nucleotides)No. ID No. #1 3′ Human glyceraldehyde-3-phosphate dehydrogenase 5NM_002046 18 #1 5′ Human eukaryotic translation elongation factor 1alpha 1 27 NM_001402 19 #2 3′ Human hydroxymethylbilane synthase 24NM_000190 20 #2 5′ Human glyceraldyhyde-3-phosphate dehydrogenase 30NM_002046 21 #3 3′ Human glyceraldehyde-3-phosphate dehydrogenase 15NM_002046 22 #3 5′ Human mitochondrial ribosomal protein L32 29NM_031903 23 #4 3′ Human β-actin 30 NM_001101 24 #4 5′ Human β-actin 29NM_001101 25 #5 3′ Human phosphoglycerate kinase 1 29 NM_000291 26 #5 5′Human phosphoglycerate kinase 1 29 NM_000291 27 #6 3′ Humanpeptidylprolyl isomerase A 29 NM_021130 28 #6 5′ Human peptidylprolylisomerase A 29 NM_021130 29 #7 3′ Human tubulin, α-1b 29 NM_006082 30 #75′ Human tubulin, β-1 29 NM_030773 31 #8 3′ Human transferrin receptor29 NM_003234 32 #8 5′ Human eukaryotic translation elongation factor 229 NM_001961 33 #9 3′ Human ubiquitin C 29 NM_021009 34 #9 5′ Humantransferrin receptor 29 NM_003234 35 #10 3′ Human TATA box bindingprotein 29 NM_003194 36 #10 5′ Human lamin B2 29 NM_032737 37 #11 3′Human α-actin, cardiac muscle 1 29 NM_005159 38 #11 5′ Human α-actin,cardiac muscle 1 29 NM_005159 39 #12 3′ Human tubulin, β-1 29 NM_03077340 #12 5′ Human tubulin, β-1 29 NM_030773 41 #13 3′ Human1-acylglycerol-3-phosphate O-acyltransferase 1 29 NM_006411 42 #13 5′Human 1-acylglycerol-3-phosphate O-acyltransferase 1 21 NM_006411 43 #143′ Human tubulin, α-1b 13 NM_006082 44 #14 5′ Humanglyceraldehyde-3-phosphate dehydrogenase 46 NM_002046 45 Human ATPsynthase, mitochondrial Fo complex subunit B1 13 NM_001688 73 Human ATPsynthase, mitochondrial Fo complex subunit B1 18 NM_001688 74 Humanpeptidylprolyl isomerase A (cyclophilin A) 18 NM_021130 75 Humanribosomal protein, large, P1 (RPLP1) 12 NM_001003 764-2. Replacement with “RNA that is not Recognized by an Innate ImmuneSystem” in 3′ Terminal and 5′ Terminal Regions of RNA Vector Genome

As shown in FIG. 2, FIG. 3 and FIG. 4, among the genome RNA sequencesconstituting the stealth RNA vector of the present invention, the 3′terminal region and the 5′ terminal region include sequence regions ofwhich function is unknown besides the essential constituents involved intranscription, replication and the like shown in the above sections 2-2.to 2-3. and so on, and these sequences include regions that can bereplaced with “RNA that is not recognized by an innate immune system”such as partial sequences of mRNA derived from House-keeping gene inplural sites.

For example, as shown in Example of FIG. 3, among the structuresexisting in 3′ terminus of native virus genome, the regions of (1) to(6) can be replaced with other non-homologous base sequences includingpartial sequences of mRNA derived from House-keeping gene in Table 1,and all of 3′ Variant 1 to 3′ Variant 6 of the stealth RNA geneexpression system are capable of achieving stable gene expression andproduction of vector particles. Also as shown in Example of FIG. 4,among the structures existing in 5′ terminus of native virus genome, theregions of (1) to (4) can be replaced with or inserted by othernon-homologous base sequence, and all of 5′ Variant 1 to 5′ Variant 5 ofthe stealth RNA gene expression system are capable of achieving stablegene expression.

It would be highly possible that the interferon inducibility is furthersuppressed by replacing sequences of these positions with “RNA that isnot recognized by an innate immune system” such as partial sequences ofmRNA derived from House-keeping gene of (Table 1).

5. Techniques for Avoiding Activation of an Innate Immune System (PAMP)by Proteins Essential for Transcription and Replication 5-1.Investigation of Value That Provides Index for PAMP Structure in GeneEncoding Protein Essential for Transcription and Replication

In Examples of the present invention, L protein (large subunit of RNApolymerase, PolL) and P protein (small subunit of RNA polymerase, PolS)of Sendai virus were selected as “RNA-dependent RNA polymerase”, Cprotein (C) of Sendai virus was selected as “protein that regulatesactivity of RNA polymerase”, and NP protein (N) of Sendai virus wasselected as “single-stranded RNA binding protein”. Although theseproteins are essential for transcription and replication from anegative-sense single-stranded RNA, it is highly possible that“pathogen-associated molecular pattern (PAMP)” exists in genome RNA ormRNA of Sendai virus encoding these proteins as shown in Non-PatentDocument 37. Therefore, it is necessary to remove a structure that is apotential PAMP from the RNA encoding these protein so as to construct astealth RNA gene expression system that does not activate an innateimmune system.

Although it is sure that active PAMPs exist in genome RNA and mRNAconstituting Sendai virus, the region where the active PAMP actuallyexists has not been elucidated. However, RNA having active PAMP musthave a structure that is clearly different from that of RNA expressed inhost cells. Thus, the present inventors first made comparison accordingto codon adaptation index (CAI) of coding region as an index in order toexamine the difference in structure between mRNA derived from an RNAvirus and mRNA of a human cell. CAI is an index for dissociation fromthe frequency of appearance of codons of mRNAs encoding 100 proteinsthat are most strongly expressed in cells of a certain biologicalspecies, and CAI=1.0 indicates that the codon use frequency is the sameas that of mRNAs of these 100 proteins (Non-Patent Document 46). As aresult of analysis according “OptimumGen Gene Design System (PatentDocument 7, GenScript USA Inc.)”, an average value of CAI of codingregions of arbitrarily selected 151 human mRNAs was 0.778, an averagevalue of CAI of seven mRNAs of Sendai virus was 0.704, and an averagevalue of CAI of seven mRNAs of measles virus belonging to the sameparamyxovirus family was 0.697, revealing that the CAI of mRNAparamyxovirus was significantly lower than the average CAI of mRNAs ofhuman cells (FIG. 5). An average value of CAI of arbitrarily selectedeleven mRNAs expressed in Escherichia coli analyzed for reference was0.698 (FIG. 5). This suggests the possibility that in use in humancells, mRNA of paramyxovirus has a structural deviation comparable tothat of mRNA of Escherichia coli which is a prokaryote, and this isrecognized as PAMP.

For examining the difference in structure between mRNA derived from RNAvirus and mRNA of human cells from other point of view, GC contents ofcoding regions were calculated. An average value of GC contents ofnative paramyxovirus-derived RNA was 47.7% to 48.5%, which wassignificantly lower than 56.3% which was an average value of GC contentsof coding regions of human mRNA (Non-Patent Document 47) (FIG. 6).Considering that genome of an RNA virus has a relatively low GC content,and adenine-rich or uridine-rich sequences have high potential to becomePAMP (Non-Patent Document 43), the GC content also has potential becomesan index suggesting the existence of PAMP.

5-2. “Codon Optimization” Application Experiment for Genes Involved inTranscription and Replication Derived from Sendai Virus

It has been confirmed that “codon optimization” for approximating suchCAI values and GC contents to average values of mRNA of human cells iseffective for disrupting a PAMP structure in a virus-derived codingregion and avoiding PAMP, as shown for HIV, SIV, hepatitis virus and thelike in the above section 3-2.

However, the above section 3-2. also indicates the result that thereplicability is largely impaired when “codon optimization” is conductedin PAMP region in a sequence of gene essential for transcription andreplication of these viruses. Therefore, it would be conventional commonknowledge that PAMP structures in sequences of genes essential fortranscription and replication highly possibly serve as secondarystructures essential for transcription and replication.

Considering various functions are generally integrated compactly invirus genome, it would be possible that a PAMP structure in a genesequence essential for transcription and replication is important forthe function of the virus also in the case of Sendai virus as is thecase with these virus genomes from the conventional findings asdescribed above. That is, it was highly expected that when codons incoding regions of proteins involved in transcription and replication,such as Sendai virus-derived “RNA-dependent RNA polymerase” for use inthe RNA gene expression system of the present invention are optimizedfor human cells, the original transcription and replication ability isalso largely impaired although PAMP can be avoided.

Under such circumstances, the present inventors dared to optimize consof all RNAs encoding proteins such as “RNA-dependent RNA polymerase” and“RNA binding protein” involved in transcription and replication forhuman cells.

In the present invention, since L protein (large subunit of RNApolymerase, PolL) and P protein (small subunit of RNA polymerase, PolS)of Sendai virus are used as “RNA-dependent RNA polymerase”, C protein(C) of Sendai virus is used as “protein that regulates activity of RNApolymerase”, and NP protein (N) of Sendai virus is used as“single-stranded RNA binding protein”, codon optimization was conductedaccording to “OptimumGen Gene Design System (Patent Document 7,GenScript USA Inc.)” which is one program general used as a codonoptimization method so as to remove PAMP from RNAs encoding theseproteins. As a result of this, CAI values fall within the range from0.86 to 0.88, and showed values approximate to those of mRNAs encodingproteins highly expressed in human cells.

Results of applying codon optimization to genes of L, P, and N proteinsof Sendai virus according to “OptimumGen Gene Design System (alsoreferred to as OGGDS method)” are shown the following (Table 2).

TABLE 2 Before optimization After optimization Codon Codon Adapta- GCAdapta- GC Gene tion content tion content name Function Index (%) Index(%) L RNA-depen- 0.68 44.0 0.88 52.5 dent RNA polymerase P Protein 0.7349.6 0.86 54.4 that regu- lates activ- ity of RNA polymerase C Protein0.73 50.1 0.88 53.5 that regu- lates activ- ity of RNA polymerase N RNAbind- 0.71 49.4 0.88 55.5 ing protein

In the above (Table 2), GC contents as well as CAI values werecalculated for RNAs after codon optimization so as to analyze theoptimized RNAs from other point of view. While GC contents of RNAsbefore optimization were in the range of 44.0% to 50.1%, GC contents ofRNAs after optimization increased to the range of 52.5% to 55.5%, aniapproximated 56.3% which is an average value of GC contents of codingregions of human mRNA (Non-Patent Document 47) (Table 2) (FIG. 6). InRNA viruses, it is known that adenine-rich or uridine-rich sequenceshave high potential to become PAMP (Non-Patent Document 36), and theexperiment result strongly suggests the possibility that the structureof virus-derived RNA approximates the structure of human mRNA by thetechnique of codon optimization, and regions having activity of PAMP areremoved at the same time.

In the present invention, an RNA vector carrying RNAs encoding NPprotein, P protein, C protein, L protein derived from Sendai virus thatare optimized for human cells by the above technique together with tenexogenous genes was constructed (FIG. 13) (Example 8), and the vectorwas expressed in Hea cells, and investigated (Example 9). It wasconfirmed that all the ten exogenous genes were expressed in adequatequantities that can be observed. Also it was confirmed that the RNAvector is capable of avoiding INF-β induction in human fibroblasts(Example 13, FIG. 17).

This reveals that the RNA vector of the present invention carrying RNAof genes that are involved in transcription and replication derived fromSendai virus and are optimized for human cells functions as an excellentstealth RNA vector having the PAMP avoiding effect.

This result also shows that any PAMP structure existing in genesessential for transcription and replication was not essential fortranscription and replication in the case of Sendai virus, and this wasan unexpected surprising result for the present inventors who dared tomade the experiment.

5-3. Investigation of Codon Optimization Method

The result of the above (Table 2) suggests that for “codon optimization”for suppressing induction of innate immune reaction by removing regionshaving active PAMP, the two numerical ranges of “CAI value” and “GCcontent” are important requirements. Thus, the present inventors plannedto conduct an experiment by applying other codon optimization method soas to investigate which one of the two requirements is more essential.As a method for codon optimization, since various methods have beenproposed as represented by GeneOptimizer Process (Non-Patent Document49) and GeneGPS Expression Optimization Technology (Patent Document 8,and Patent Document 9) besides the aforementioned OGGDS method, it ispossible to confirm that the equivalent effect is achieved when a methodother than the aforementioned OGGDS method is applied.

Thus, a codon optimization method based on GeneGPS ExpressionOptimization Technology (hereinafter, also referred to as a GGEOTmethod) which is a generally used “codon optimization” techniquelikewise the OGGDS method was applied to a template DNA encoding NPprotein, P protein, C protein, and L protein of Sendai virus, and an RNAvector capable of carrying ten exogenous genes (FIG. 15) was prepared inthe same manner. By the verification by the method of Example 9, it wasconfirmed that the stealth RNA vector was a stealth RNA vector capableof avoiding induction of the innate immune reaction as with the stealthRNA vector optimized by the OGGDS method (data not shown).

Optimization by the OGGDS method and optimization by the GGEOT methoduse completely different algorithms, and the identity between the basesequences of nucleic acid optimized by these two methods was 77% to 80%,revealing that considerably different nucleotides were e ected for codonoptimization (Table 4).

The foregoing demonstrated that the method for optimizing the genesencoding “RNA-dependent RNA polymerase”, “protein that regulatesactivity of RNA polymerase”, and “single-stranded RNA binding protein”for preparing a stealth RNA gene expression system does not rely on aspecific codon optimization method, and any codon optimization methodbased on any algorithm can be applied as a codon optimization method ofthe present invention.

The following (Table 3) shows values of GC contents and CAI values aftercodon optimization by the GGEOT method for L, P, C and N protein genesof Sendai virus, in comparison with the values by the OGGDS method shownin the above (Table 2). Since the GGEOT method lacks a calculationprogram for “CAI value”, the calculation was conducted according to thecalculation program for “CAI value” of the OGGDS method.

Also (Table 4) shows the original sequence, and a value of homology(identity) between the sequences after application of OGGDS and thesequences after application of GGEOT for each of L, P, C and N proteingenes.

TABLE 3 After optimization After optimization (OptimumGen Gene (GeneGPSExpression Before optimization Design System) Optimization Technology)Codon Codon Codon Gene Adaptation GC content Adaptation GC contentAdaptation GC content name Function Index (%) Index (%) Index (%) LRNA-dependent 0.68 44.0 0.88 52.5 (0.71) 51.1 RNA polymerase P Proteinthat regulates 0.73 49.6 0.86 54.4 (0.70) 59.9 activity of RNApolymerase C Protein that regulates 0.73 50.1 0.88 53.5 (0.72) 51.4activity of RNA polymerase N RNA binding protein 0.71 49.4 0.88 55.5(0.70) 59.4

TABLE 4 N (NP) gene Native Virus 75.94% Optimized with Genome OGGDSMethod Native Virus 76.13% Optimized with Genome GGEOT Method Optimizedwith 80.38% Optimized with OGGDS Method GGEOT Method PolS (P) geneNative Virus 74.17% Optimized with Genome OGGDS Method Native Virus74.99% Optimized with Genome GGEOT Method Optimized with 78.09%Optimized with OGGDS Method GGEOT Method C gene Native Virus 77.24%Optimized with Genome OGGDS Method Native Virus 76.91% Optimized withGenome GGEOT Method Optimized with 77.56% Optimized with OGGDS MethodGGEOT Method GENE A Homology GENE B between GENE A & B PolL (L) geneNative Virus 75.31% Optimized with Genome OGGDS Method Native Virus74.92% Optimized with Genome GGEOT Method Optimized with 76.72%Optimized with OGGDS Method GGEOT Method

5-4. Essential Index for “Codon Optimization”

While CAI is used as an index for estimating the translation efficiencyof mRNA in human cells (Non-Patent Document 46), codon optimization isused as a measure for eliminating a structure having active RAMP from avirus-derived RNA in the present invention, and elevation in thetranslation efficiency may not be necessarily obtained.

While CAI is an “index for dissociation from the frequency of appearanceof codons mRNAs encoding 100 proteins that are most strongly expressedin cells of a certain biological species”, an objective standard forselecting 100 proteins which forms a standard has not been shown.Optimization by the OGGDS method and optimization by the GGEOT methodthat could achieve expression of ten genes while suppressing inductionof the equivalent innate immune reaction in human-derived culture cellsin the present invention were compared from each other, and the CAIvalue of the latter case (calculated by the OGGDS method) did notsignificantly vary before optimization and after optimization (Table 3).

Meanwhile, the GC content was 51% or more, and about 60% at mostregardless of the employed optimization method. This shows that GCcontent is more effective than CAI value as an index of the possibilitythat a structure having active PAMP has been removed. In other words, asan index in “codon optimization” for “stealth RNA gene expressionsystem” in which induction of the innate immune reaction is suppressed,GC content is the most excellent index, and the possibility that thestructure having active PAMP has been removed is estimated if the GCcontent after codon optimization of the virus-derived protein is atleast 50.0% or more, desirably 52.0% or more.

From the above, the wording “codon optimization” for an RNA geneexpression system used in the present invention means adjusting all ofthe base sequences encoding proteins required for the RNA geneexpression system to have a GC content of 50 to 60%, preferably 52 to56%.

As a result of modification of the base sequence by codon optimization,the sequences encoding C protein and V protein that had existed in Pgene region disappeared, and neither C protein nor V protein wasexpressed from optimized P gene. The RNAs encoding C protein and Vprotein are not essential because gene expression is conducted even whenthe RNAs are completely removed, however, in the case of C protein, inparticular, it is preferred to add C protein gene RNA that is optimizedas described above into the sequence because protein is important forproperly regulating the expression amount of the RNA vector of thepresent invention. Also V protein RNA can be added into the sequence asnecessary after it appropriately undergoes similar codon optimization.

6. Method for Carrying a Large Number of Exogenous Genes on Stealth RNAin the Present Invention (Transcription Cassette Linking Method) 6-1.Investigation of Method for Carrying Six or More Genes on One Vector

Next, the method for carrying six or more, for example, ten exogenousgenes on a stealth RNA in a simple manner was investigated. Since an RNAmolecule itself cannot be engineered by gene recombination technique,every construction including carrying of an optimized virus-derived genein 5-4. was conducted as a cDNA, and RNA was prepared by DNA-dependentRNA polymerase such as T7 RNA polymerase derived from T7 phage using thecDNA as a template.

For preparing a DNA molecule carrying ten genes in the simplest manner,a method of introducing restriction endonuclease cleavage sites peculiarto each gene upstream and downstream the respective cDNA of each gene,and inserting the cDNAs cleaved by restriction endonucleases intosequence is conceivable. However, this method is impractical because atleast 20 different restriction endonucleases are required, and it isnecessary to prepare every cDNA again for changing the combination ofgenes or for changing the position on the stealth RNA.

6-2. Preparation of “Tandem Transcription Cassette” Carrying Two Genes

In the present invention, as described in the above section 2-1. (FIG.1), the technique of preparing “tandem transcription cassette” carryingtwo genes, and linking plural tandem transcription cassettes isemployed. In this technique, genes to be installed were designed to havethe same structure, and designed so that they can be installed at anyposition in the stealth RNA (FIG. 7). In this designing method,restriction endonuclease cleavage sites are separately provided:restriction endonuclease A cleavage site on 5′ upstream side of the geneto be installed, and restriction endonuclease B cleavage site on 3′downstream side of the gene, and cDNA cleaved at these sites is insertedinto a DNA molecule which is to be a template. The template DNA intowhich the cDNA is to be inserted is provided with recognition sites byrestriction endonuclease C and restriction endonuclease D, in additionto recognition sites by restriction endonuclease A and restrictionendonuclease B. These combinations of restriction endonucleases areselected so that the DNA fragment cleaved by restriction endonuclease Acan covalently bind with the DNA fragment cleaved by restrictionendonuclease D, and the DNA fragment cleaved by restriction endonucleaseB can covalently bind with the DNA fragment cleaved by restrictionendonuclease C. There are a large number of such combinations, besidesthe combination Acc65I and BsiWI and the combination of XhoI and SalIrecited in Examples, combination of XbaI, and SpeI NheI, and combinationof BamHI and BglII are conceivable. In this case, any cDNA to beinstalled is structurally restricted to design so that recognition sitesby restriction endonuclease A, restriction endonuclease B, restrictionendonuclease C, and restriction endonuclease D are absent within itssequence. By using such combinations of restriction endonucleases, DNAfragments each carrying two cDNAs are first prepared (FIG. 8).

6-3. “Transcription Cassette” Linking Method

Next, five DNA fragments each carrying two cDNAs linked in this mannerare connected to prepare a DNA carrying a total of ten cDNAs (FIG. 7,FIG. 8 and FIG. 9). In Examples, a DNA fragment carrying two linkedcDNAs prepared in the above section 6-2. was cleaved a restrictionendonuclease called SapI and isolated. The protruding end structure ofthe DNA cleaved by SapI has such a structure that three nucleotides areprotruded on 5′ side, and by setting the sequence of three nucleotidesarbitrarily, 4×4×4=64 patterns of protruding end structure can beselected (FIG. 7) (FIG. 9). Therefore, it is possible to bind five DNAfragments accurately as designed and to collect them as me DNA molecule(FIG. 9). In this case, cDNAs of genes to be installed are designed sothat recognition site by SapI, in addition to recognition sites byrestriction endonuclease A, restriction endonuclease B, restrictionendonuclease C, and restriction endonuclease D are absent within itssequence (FIG. 7).

The restriction endonuclease having the characteristic of generating anygiven single-stranded protruding end structure in the sequencerepresented by NN or NNN on the terminus generated by digestion is notlimited to SapI, and the equivalent results are obtained with variousrestriction endonucleases including BbsI, BbvI, BcoDI, BfuAI, BsaI,BsmBI, BsmFI, BtgZI, EarI, FokI, HgaI, and SfaNI. The equivalent effectcan be obtained also with AlwNI, BglI, BstAPI, BstXI, DraIII, SfiI andthe like having an indefinite sequence in the recognition site. Thisstep is not necessarily cloning by a restriction endonuclease, and amethod using homologous recombination (In-Fusion HD Cloning System(TAKARA-Bio, Inc) or Gibson Assembly System (New England Biolabs, Inc))can also be employed. The step of incorporating into a circular plasmidDNA after connecting ten cDNAs can be achieved also by covalent bondingusing ordinary T4 DNA ligase without using the method of incorporatinginto pDONR-221 or the like by homologous recombination (Gateway System(Life Technologies, Inc.)) shown in Examples. Also it is possible toprepare a DNA molecule carrying any of one to ten genes by the methodshown in FIG. 9.

6-4. Regulation of Expression Level of Exogenous Gene

Generally, in the case of inserting plural exogenous genes in anegative-sense single-stranded RNA gene expression system containinggenes respectively encoding a set of RNA-dependent RNA polymerase (PolSand PolL), single-stranded RNA binding protein (N), and RNA polymeraseactivity regulating protein (C), it is known that the expression levelis higher as the position is closer to upstream 3′ terminal site. Thestealth RNA gene expression system of the present invention also showsthis trend. In the present invention since a large number of exogenousgenes can be incorporated in any order in the manner of integratingcassettes, the genes can be conveniently arranged in the order of thedesired expression levels. Also the expression levels of the proteinsgenerated from respective genes can be regulated by changing thetranslation efficiency (FIG. 20).

7. Synthesis of Stealth RNA

Next, the DNA fragment in which ten cDNAs are linked, prepared in theabove section 6., and a gene encoding single-stranded RNA bindingprotein (hN), a gene encoding protein that regulates activity of RNApolymerase (hC), and genes encoding an RNA-dependent RNA polymerase(hPolL and hPolS) having codons optimized for human cells in the manneras described in the above section 5. (hereinafter, also referred to“humanized”, and “h” is added to the abbreviated name of the protein)were linked to prepare a circular template cDNA for synthesizing astealth RNA (FIG. 10). The structure of the stealth RNA can be selectedfrom a negative strand and a positive strand depending on the positionof the promoter recognized by the RNA polymerase. Here, the RNA havingthe same orientation as the mRNA expressed from the gene installed onthe stealth RNA is defined as a positive strand, and the RNA having theorientation complementary to the mRNA is defined as a negative strand,and FIG. 10 illustrates preparation of a template for synthesizing anegative-sense RNA by using T7 RNA polymerase. Downstream the T7promoter (Non-Patent Document 50), a ribozyme derived from antigenome ofhuman hepatitis D virus for cleaving RNA and creating an accurate end(Non-Patent Document 51) and a transcription termination signal of T7RNA polymerase (Non-Patent Document 50) are arranged so that RNAcorresponding to the entire length of the stealth RNA can besynthesized. The enzyme used for synthesis of RNA is not limited to T7RNA polymerase, but any DNA-dependent RNA polymerase that can be used inEscherichia coli or animal cells can be used. For example, T3 RNApolymerase derived from Escherichia coli T3 phage (Non-Patent Document52) and SP6 RNA polymerase derived from salmonella SP6 phage (Non-PatentDocument 53) can also be used in combination with the promoter and thetranscription termination point recognized by these enzymes. Theribozyme is used for accurately cleaving 3′ terminus of RNA, and notonly the ribozyme derived from antigenome of human hepatitis D virus asused in Examples, but also a ribozyme derived from genome of humanhepatitis D virus (Non-Patent Document 51), a hairpin ribozyme oftobacco ringspot virus (Non-Patent Document 54), and a short inhibitoryRNA (siRNA) capable of cleaving RNA in cells (Patent Document 55) can beused.

The cDNA complementary to the entire length of the stealth RNA cDNA iscloned into a plasmid having a replication origin derived from p15A(Non-Patent Document 56). Since the plasmid having a replication originderived from p15A is maintained in a low copy number state inEscherichia coli, not only it is advantageous for stably retaining alarge DNA fragment in Escherichia coli, but also it can coexist inEscherichia coli with a plasmid for expressing N protein having areplication origin derived from ColE1 in the method 2 for reconstitutingthe stealth RNA gene expression system (Non-Patent Document 56). InExamples, the plasmid having a replication or derived from p15A carriesampicillin resistance, and the plasmid having a replication originderived from ColE1 carries kanamycin resistance, and the two plasmidsare maintained in the same Escherichia coli by double selection ofampicillin and kanamycin, however, the combination of antibiotics is notlimited to this example. Regarding the combination of plasmids, aplasmid having a replication origin derived from F factor in place ofthe replication origin derived from p15A and a plasmid having areplication origin derived from pUC in place of the replication originderived from ColE1 can be used.

8. Reconstruction of Stealth RNA Gene Expression System 8-1.Conventional Reconstruction Method

Reconstruction of a stealth RNA gene expression system composed of anegative-sense single-stranded RNA and a protein that binds to the RNAcan be achieved in two methods. The first method is a technique using avirus having genome of a negative-sense single-stranded RNA, and knownas a vector reconstituting method using the virus, wherein apositive-sense single-stranded RNA complementary to the negative sensesingle-stranded RNA is expressed in animal cells by using T7 RNApolymerase, and simultaneously, three proteins, NP (N), P (PolS), and L(PolL) are expressed in the cells, and thus a stealth RNA geneexpression system of a positive-sense RNA is reconstituted (FIG. 11)(Non-Patent Document 57, and Patent Document 3). The merit of thismethod lies in the convenience that by using animal cells expressing T7RNA polymerase stably, reconstitution can be achieved only byintroducing the plasmid DNA which is a material into the cells. On theother hand, it is also known that since a plasmid containing a templatecDNA for synthesizing a positive-sense single-stranded RNA, and threeplasmids carrying genes for expressing three proteins, NP, P, and L aresimultaneously introduced into cells, gene recombination often occursamong these DNA molecules, and mutation is inserted into the structureof the negative-sense single-stranded RNA to be prepared (Non-PatentDocument 57). In Examples of Patent Document 3, plasmids for expressingM, F, and HN proteins are further added so as to increase the efficiencyof reconstitution.

8-2. Reconstruction Method Developed in the Present Invention

In the second method, first, a complex of a negative-sensesingle-stranded RNA and a NP protein (N) having a single-stranded RNAbinding ability is prepared in Escherichia coli, and the complex isintroduced into animal cells in which P (PolS) protein and L (PolL)protein are expressed, to reconstitute a stealth RNA gene expressionsystem (FIG. 12). In this method, first, mRNAs encoding N protein and astealth RNA are synthesized by T7 RNA polymerase respectively from twoplasmids that can coexist in Escherichia coli, and the single-strandedRNA binding protein (N) and the stealth RNA are co-expressed inEscherichia coli to produce a complex. Although the method ofreconstituting an RNA virus by using an RNA-protein complex isolatedfrom a naturally occurring RNA virus as a material is disclosed inNon-Patent Document 58, the method developed in the present inventionenables reconstitution using a stealth RNA synthesized by generecombination techniques as a material. Although this methoddisadvantageously requires a larger number of processes and is morecomplicated as compared with the first method, this method makes itpossible to reconstitute a stealth RNA gene expression system withoutintroducing mutation into genome RNA using Escherichia coli in which agene involved in homologous recombination (RecA) and a gene encodingRNase (RNaseE) are disrupted (Non-Patent Document 59).

That is, the method for reconstituting a stealth RNA gene expressionsystem developed in the present invention is a method of preparing acomplex of a negative-sense single-stranded RNA and a protein having asingle-stranded RNA bindability (for example, NP protein (N)) in hostcells expressing T7 RNA polymerase in advance, and introducing thecomplex into animal cells in which the RNA-dependent RNA polymerase (forexample, P (PolS) protein and L (PolL) protein) is expressed toreconstitute a stealth RNA gene expression system. Preferably as hostcells, Escherichia coli in which RecA gene and RNaseE gene are disruptedand T7 RNA polymerase is expressed is used.

Subsequently, using the stealth RNA carrying ten genes synthesized bythe method shown in the above section 7., a stealth RNA gene expressionsystem is constructed by any of the methods described in the abovesection 8. (FIG. 13). It was confirmed that the gene expression systemhaving a negative-sense single-stranded RNA prepared in this manner canpersistently express all the ten installed genes by stable expression ofthree drug resistance characters (puromycin resistance, Zeocinresistance, and hygromycin resistance), four fluorescent proteins (EGFP,E2-Crimson, EBFP2, and Keima-Red), and three luciferases (fireflyluciferase, Renilla luciferase, and Cypridina noctiluca luciferase)(FIG. 14).

8-3. Order of Linking RNA-Binding Protein (hN, hC, hPol) Genes inStealth RNA Gene Expression System

In the stealth RNA gene expression system of the present invention, thepositions on the stealth RNA of the gene encoding single-stranded RNAbinding protein (hN), the gene encoding protein that regulates activityof RNA polymerase (hC), and the gene encoding an RNA-dependent RNApolymerase (hPolS) are not limited to the order of hN-hC-hPolS from 3′terminal side shown in FIG. 1 For example, a stealth RNA gene expressionsystem can be constructed while the order is changed as is hN-hPolS-hCor hPolS-hN-hC (FIG. 16).

8-4. Need for Mutation in Virus-Derived Protein Genes in Stealth RNAGene Expression System

In the stealth RNA gene expression system, there is no need of existenceof specific mutation in the proteins expressed from the humanized geneencoding single-stranded RNA binding protein (hN), the humanized geneencoding protein that regulates activity of RNA polymerase (hC), and thehumanized genes encoding an RNA-dependent RNA polymerase (hPolS, hPolL).

As described in the above section 3-3., in the conventional technique,introduction of a mutation for suppressing PAMP activity into thevirus-derived RNA-dependent RNA polymerase or the like has been used asthe most effective means for avoiding the innate immune system.

However in the present invention, since any active PAMP is removed fromthe virus-derived protein gene by codon optimization according to themethod shown in the above section 5., it is not necessary topreliminarily introduce a mutation into a virus-derived protein in aprotein level. For example, even when genes expressing NP, P, C and Lproteins derived from Sendai virus Z strain which is a wild-typeparamyxovirus known to have strong interferon inducibility are used,they can be used as a material for the stealth RNA gene expressionsystem through optimization by the method shown in the above section 5.For example, as the gene that expresses L protein shown as “hPol” in(FIG. 16), a gene sequence derived from Z strain is optimized for humancells and used.

9. Verification of Activity of Inducing Innate Immune System

9-1. Comparison with Innate Immune System Avoiding Effect inConventional Art

Next, for comparing the activity of inducing the innate immune system ingene introduction between the stealth RNA vector carrying the stealthRNA gene expression system and a conventional art, a stealth RNA vectorcarrying exactly the same four genes (Keima-Red, Blasticidin S resistantgene, EGFP, and Kusabira-Orange) as the persistent expression typeSendai virus vector which is a conventional art described in FIG. 1B ofNon-Patent Document 7 was prepared. Genes were introduced into humanprimary culture fibroblasts using these two vectors, and the amount ofinterferon beta mRNA after 24 hours was quantified by the Real-Time PCRmethod (FIG. 17). Induction by the stealth RNA vector was within fivetimes the amount of interferon mRNA in normal cells although it does notcarry V gene that suppresses the innate immune system. On the otherhand, in the conventional art, induction of 47 times compared withnormal cells was observed although V gene is contained (FIG. 17). Thisresults reveal that in the stealth RNA gene expression system,activation of the innate immune system could be avoided even under thecondition where a factor that inhibits the innate immune system isabsent.

9-2. Further Avoidance of Activation of Innate Immune System

Activity of inducing the innate immune system is also influenced by thekind of the cells retaining the stealth RNA gene expression system, orthe strength of gene expression from the stealth RNA gene expressionsystem. For example, interferon beta is little induced in human-derivedHeLa cells, while it is strongly induced in human-derived 293 cells. Asthe gene expression is strengthened for production ofbiopharmaceuticals, the induction of interferon beta is strengthened. Inthe case of production of biopharmaceuticals using a stealth RNA geneexpression system, since mutation of RNA genome (Non-Patent Document 39)by activity of cytoplasmic adenosine deaminase induced by interferon(Adenosine deaminase acting on RNA, ADAR1) is problematic, it is desiredto further suppress the innate immune system inducing activity remainingin the stealth RNA gene expression system.

This object can be achieved by addition carrying a factor thatsuppresses the innate immune system on the stealth RNA gene expressionsystem (FIG. 18 and FIG. 19). As such a factor, a deletion mutant of“molecular pattern characteristic of pathogenic microorganism(Pathogen-associated molecular pattern, PAMP)” receptor RIG-I existingin cytoplasm (RIG-IC) (Non-Patent Document 71), C-terminal region ofSendai virus V protein (Non-Patent Document 72), and PSMA7 which is aconstituent of proteasome (Non-Patent Document 73) can be recited.

10. Regulation of Gene Expression Level

Next, how the level of gene expression in the stealth RNA geneexpression system varies by regulating the expression of factorsinvolved in transcription and replication installed on the vector wasexamined (FIG. 20, FIG. 21). FIG. 20 indicates positive-sense RNAsequences. Expression of each factor can be regulated by altering theefficiency of translation from mRNA to protein. The simplest means formodifying the translation efficiency is to change the 5′ noncodingsequence directly in front of the translation initiation codon (AUG). Itis considered that the highest translation efficiency in animal cells isachieved when the sequence directly in front of AUG is 5′-CCACC-3′ (SEQID NO: 18) (Non-Patent Document 60). On the other hand, it is possibleto reduce the translation efficiency by inserting a short coding regionon 5′ upstream side (Non-Patent Document 61). In Examples, a vector inwhich expressions of single-stranded RNA binding protein aim and proteinthat regulates activity of RNA polymerase (hC) are suppressed to 40% and23%, respectively while expression of RNA-dependent RNA polymerase(hPolS and hPolL) is kept constant was prepared, and expressions offirefly luciferase installed thereon were compared (FIG. 20). Expressionof the installed luciferase gene increased by suppressing the expressionof hN or hC, and increase in gene expression of up to 79 times wasobserved by combining expression suppression of hN and expressionsuppression of hC.

Regulation of gene expression level as described above can be conductedonly by regulation of the expression level of the protein that regulatesactivity of RNA polymerase (hC) (FIG. 21). In this case, the stealth RNAgene expression system can be reconstructed even when hC gene isdeleted, and the gene expression level is maximum, and hence, hC gene isnot an essential element for the stealth RNA gene expression system.However, proliferation of cells will be strongly inhibited when theexpression of the installed gene is too strong, and hence it ispractical to realize gene expression adapted to the purpose byexpressing hC protein at an appropriate level.

As important characteristics in the gene expression system, selectivityof optimum expression level depending on the purpose is recited. Forexample, in cell-reprogramming, cell death is induced when theexpression of the transcription factors is too strong. In production ofbiopharmaceuticals, the production efficiency is deteriorated when theexpression is weak. Generally, it is difficult to alter the expressionlevel of the vector in a gene expression system using an RNA virus. Incontrast, in the stealth RNA gene expression system, it is possible toalter the strength of the expression freely depending on the use purposeby finely regulating the expression balance of the individualconstituents.

Next, an attempt was made to prepare a vector particle capable ofintroducing the stealth RNA gene expression system into various animalcells by enclosing therein the stealth RNA gene expression systemcompleted through the processes described above. When three proteins M,F, and HN of paramyxovirus were expressed in BHK cells having thestealth RNA gene expression system in cytoplasm by using a strong SRαpromoter, vector particles having gene introduction activity weredetected in the culture supernatant of the cells. The infectivity titerwas about 10⁷ infectious units/mL, and high activity comparable to thatby a conventional persistent expression type Sendai virus vector wasobtained. This vector particle adsorbs to the cell surface by theactivities of F and HN proteins, and is capable of introducing thecontent, namely, the stealth RNA gene expression system into thecytoplasm by the fusion of membranes. Since this process does notrequire cell division, the gene can be introduced into nondividingcells.

While the cell specificity and the species specificity of the cells forwhich gene introduction can be made are determined by the origin of Fand HN proteins, genes could be introduced into a very wide range ofhuman cells and animal cells including blood cells of peripheral bloodwhen F and HN proteins of Sendai virus were used.

11. Removal of Vector

In a persistent expression type Sendai virus vector which is aconventional art, rapid vector removal is successfully achieved bysuppressing the activity of RNA-dependent RNA polymerase by siRNA(Patent Document 3, and Non-Patent Document 7). Thus, whether a vectorin the stealth RNA gene expression system can be removed by a similarmethod was examined. (FIG. 23). Since the base sequence of humanizedRNA-dependent RNA polymerase (hPolL) possessed by the stealth RNA geneexpression system is different from that in the persistent expressiontype Sendai virus vector which is a conventional art, three siRNAs werenewly synthesized, and the activities thereof were examined. Removalcould be achieved in the same manner as in the conventional art by usingone of the three siRNAs (target sequence is SEQ ID NO: 46) (FIG. 23).Thus, it was found that for the stealth RNA gene expression system ofthe present invention, the vector removing method by RNAi that has beenused in a conventional persistent expression type Sendai virus vectorcan be applied. Likewise, the removing method using microRNA (miRNA) isalso applicable, and as described, for example, in Patent Document 3,removal can be achieved by reaction with endogenous miRNA by inserting atarget sequence of microRNA (miRNA) into the 3′ noncoding region or the5′ noncoding region of the exogenous gene.

12. Use of Stealth RNA Expression System of the Present Invention

The negative-sense single-stranded stealth RNA vector used in thestealth RNA expression system of the present invention can carry six ormore, further up to ten any given genes such as human-derived genes, andcan carry a length of 5,000 nucleotides, further a length of up to15,000 nucleotides.

Since the system is stealthy, namely the system is capable of avoidingactivation of an innate immune system in animal cells such as humancells, and removal of the vector can be easily conducted, a wide varietyof uses including cell-reprogramming technology requiring simultaneousintroduction of plural genes, gene therapy including giant gene,regenerative medicine, production of biopharmaceuticals and the like areconceivable.

Specifically, the following embodiments are conceivable. (1) Applicationto the technique of preparing iPS cells of high quality for clinical usein regenerative medicine with high efficiency

When six or more genes for reprogramming animal cells such as humancells, for example, a total of six genes including four Yamanaka factors(KLF4, OCT4, SO X2 and c-Myc)+BRG1+BAF155 for converting into iPS cellsare installed, the length amounts to 13,132 nucleotides. When six genes,OCT4, KLF4, SOX2, c-MYC, NANOG, and LIN28 are installed, the lengthamounts to 7,000 nucleotides.

Actually, these six genes were installed on the stealth RNA vector ofthe present invention (FIG. 25), and expressed in human embryonicfibroblasts, and initialization efficiency exceeding 40% was achieved(Example 21). It has been confirmed that the order of four Yamanakafactors (KLF4, OCT4, SOX2 and c-Myc) to be installed on in this case canbe appropriately changed (data not shown).

A similar experiment was conducted in the absence of animal components(Xeno-free) and feeder cells (Feeder-free) by using human peripheralblood cells as a material, and as a result, higher initialization thanthe conventional method could be conducted (data not shown).

Also by carrying the four genes, KLF4, OCT4, SOX2, and c-MYC, and CHD1gene encoding a chromatin remodeling factor (a total of 9,907 nucleotidelength), and further adding TET1 gene encoding DNA demethylase (a totalof 11,203 nucleotide length), it is possible to increase theinitialization efficiency.

As other possible combinations, by expressing a total of eight genesincluding further added two oocyte-specific histones in human somaticcells, it is possible to prepare human iPS cells efficiently.

(2) Application to Regenerative Medicine Utilizing Direct ReprogrammingTechnology for Creating Useful Cells of Nerve Cells, Neural Stem Cells,Stem Cells, Pancreatic Beta Cells and the Like from Human Tissue Cells(Blood, Skin, Placenta and the Like)

For example, in the technique of reprogramming human fibroblasts intomotor nerves, three genes, HB9, ISL1, and NGN2 can be added to fourgenes, LHX3, ASCL1, BRN2, and MYT1L, and a total of seven genes (9, 887nucleotide length) can be installed.

(3) Production of Biopharmaceuticals Made Up of Plural Subunits

It is useful for producing immunoglobulins G, and M because the genescorrespond thereto are giant, and the subunits are required to beexpressed simultaneously in the same cell, and regulation of theexpression amount of each subunit is required.

Actually an H (μ) chain gene, an L (κ, λ) chain gene and a J gene ofhuman immunoglobulin were installed on the stealth RNA vector of thepresent invention (FIG. 24), and human immunoglobulin M was produced byusing BHK cells (Example 22). In that case, the present inventors alsosucceeded in expressing H chain, L chain, and μ chain in a ratio ofroughly 1:1:0.2 by considering the order in which the genes areinstalled.

The present inventors also succeeded in expressing human bispecificantibody by carrying four cDNAs of human immunoglobulin (two H chainsand two L chains) on the stealth RNA vector of the present invention(Example 23).

(4) Application to Expression of Drug-Discovery Target Protein Made Upof Plural Subunits

For example, by carrying six subunits, gp91phox, p22phox, Rac, p47phox,p67phox, and p40phox on the stealth RNA vector of the present invention,and expressing them simultaneously, it is possible to express NADPHoxidase of the drug-discovery target enzyme (Nox2).

(5) Use as Gene Therapy Vector for Disease for Which Responsible gene isgiant gene, by carrying the giant gene on stealth RNA vector of thepresent invention and expressing it persistently

Specifically, cDNA of blood coagulation factor VIII which is a productof gene responsible for hemophilia A (7053 nucleotide length) and cDNAof dystrophin which is a product of gene responsible for Duchennemuscular dystrophy (11058 nucleotide length) can be used while they areinstalled on the stealth RNA vector of the present invention (FIG. 24).

EXAMPLES

Hereinafter, the present invention will be described more specificallyby way of Examples, however, it is to be noted that the presentinvention is not limited to these Examples.

Other terms and concepts in the present invention are based on themeanings of the terms that are commonly used in the art, and varioustechniques used for carrying out the present invention can be carriedout easily and securely by a person skilled in the art according knownliterature and the like except for the techniques of which source isparticularly specified. Various analyses were conducted according to themethods described in the instruction manuals, catalogues and the like ofthe employed analytical instruments, reagents or kits.

The contents described in the conventional art literature, patentpublications, patent application specifications cited in the presentdescription are referred to as if they were described in the presentinvention.

Example 1 Preparation of DNA Fragments Carrying Ten Exogenous Genes (1)

The following genes were amplified by PCR to have a structure ofAcc65I-cDNA-XhoI and sub-cloned (FIG. 7).

1) Firefly luciferase: (GenBank Accession Number AY738224)

2) Renilla luciferase: (GenBank Accession Number AY738228)

3) Enhanced Green Fluorescent Protein (EGFP): (GenBank Accession NumberU55761)

4) Puromycin resistant gene (synthesized while codons were optimized forhuman cells): Non-Patent Document 62, SEQ ID NO: 47

5) Cypridina noctiluca luciferase: Non-Patent Document 63 (GenBankAccession Number AB177531)

6) E2-Crimson: derived from pE2-Crimson (Clontech Laboratories, Inc),SEQ ID NO: 48

7) Enhanced Blue Fluorescent Protein 2 (EBFP2): Non-Patent Document 64(GenBank Accession Number EF517318)

8) Zeocin resistant gene (synthesized while codons were optimized forhuman cells): Non-Patent Document 65, SEQ ID NO: 49

9) dKeima-Red: Non-Patent Document 66 (GenBank Accession NumberAB209968)

10) Hygromycin B resistant gene (synthesized while codons were optimizedfor human cells): Non-Patent Document 67, SEQ ID NO: 50

Example 2 Preparation of DNA Fragment Carrying Ten Exogenous Genes (2)

Next, the following plasmids were prepared.

All the nucleic acids used in the present Example are DNA fragments, anda sequence specified as a negative-sense RNA sequence in the sequencinglistings such as SEQ ID NO: 1 or SEQ ID NO: 4 means a corresponding DNAsequence. This also applies to other Examples using a DNA fragment.

1) Plasmid #1

Between the ApaI cleavage site and the StuI cleavage site of plasmidLITMUS38i (New England BioLab, Inc), a DNA having the followingstructure is cloned: SapI cleavage site-attB1 (SEQ ID NO: 51)-SEQ ID NO:1-SEQ ID NO: 24-Acc65I cleavage site-SalI cleavage site-SEQ ID NO:25-SEQ ID NO: 4-ctt-SEQ ID NO: 1-SEQ ID NO: 26-BsiWI cleavage site-XhoIcleavage site-SEQ ID NO: 27-SEQ ID NO: 4-SapI cleavage site

2) Plasmid #2

Between the ApaI cleavage site and the StuI cleavage site of plasmidLITMUS38i, a DNA having the following structure is cloned: SapI cleavagesite-SEQ ID NO: 1-SEQ ID NO: 28-Acc65I cleavage site-SalI cleavagesite-SEQ ID NO: 29-SEQ ID NO: 4-ctt-SEQ ID NO: 1-SEQ ID NO: 30-BsiWIcleavage site-XhoI cleavage site-SEQ ID NO: 31-SEQ ID NO: 4-SapIcleavage site

3) Plasmid #3

Between the ApaI cleavage site and the StuI cleavage site of plasmidLITMUS38i, a DNA having the following structure is cloned: SapI cleavagesite-SEQ ID NO: 1-SEQ ID NO: 32-Acc65I cleavage site-SalI cleavagesite-SEQ ID NO: 33-SEQ ID NO: 4-ctt-SEQ ID NO: 1-SEQ ID NO: 35-SEQ IDNO: 4-SapI cleavage site

4) Plasmid #4

Between the ApaI cleavage site and the StuI cleavage site of plasmidLITMUS38i, a DNA having the following structure is cloned: SapI cleavagesite-SEQ ID NO: 1-SEQ ID NO: 36-Acc65I cleavage site-SalI cleavagesite-SEQ ID NO: 37-SEQ ID NO: 4-ctt-SEQ ID NO: 1-SEQ ID NO: 38-BsiWIcleavage site-XhoI cleavage site-SEQ ID NO: 39-SEQ ID NO: 4-SapIcleavage site

5) Plasmid #5

Between the ApaI cleavage site and the StuI cleavage site of plasmidLITMUS38i, a DNA having the following structure is cloned: SapI cleavagesite-SEQ ID NO: 1-SEQ ID NO: 36-Acc65I cleavage site-SalI cleavagesite-SEQ ID NO: 37-SEQ ID NO: 4-ctt-SEQ ID NO: 1-SEQ ID NO: 38-BsiWIcleavage site-XhoI cleavage site-SEQ ID NO: 39-SEQ ID NO: 4-attB2 (SEQID NO: 52)-SapI cleavage site

Example 3 Preparation of DNA Fragments Carrying Ten Exogenous Genes(3)(see FIG. 8)

Next, the following Plasmids were prepared.

1) Plasmid #1C

Between Acc65I-SalI of plasmid #1, an Acc65I-XhoI fragment containingfirefly luciferase gene was cloned to prepare plasmid #1B. Further,between BsiWI-XhoI of plasmid #1B , a Acc65I-XhoI fragment containingRenilla luciferase gene was cloned to prepare plasmid #1C.

2) Plasmid #2C

Between Acc65I-SalI plasmid #2, an Acc65I-XhoI fragment containing EGFPgene was cloned to prepare plasmid #2B. Further, between BsiWI-XhoI ofplasmid #2B, an Acc65I-XhoI fragment containing puromycin resistant genewas cloned to prepare plasmid #2C.

3) Plasmid #3C

Between Acc65I-SalI of plasmid #3, an Acc65I-XhoI fragment containingCypridina noctiluca luciferase gene was cloned to prepare plasmid #3B.Further, between BsiWI-XhoI with plasmid #3B, an Acc65I-XhoI fragmentcontaining E2-Crimson gene was cloned to prepare plasmid #3C.

4) Plasmid #4C

Between Acc.65I-SalI of plasmid #4, an Acc65I-XhoI fragment containingEBFP2 gene was cloned to prepare plasmid #4B. Further, betweenBsiWI-XhoI of plasmid #4B, an Acc65I-XhoI fragment containing Zeocinresistant gene was cloned to prepare plasmid #4C.

5) Plasmid #5C

Between Acc65I-SalI of plasmid #5, an Acc65I-XhoI fragment containingdKeima-Red gene was cloned to prepare plasmid #5B. Further, betweenBsiWI-XhoI of plasmid #5B, an Acc65I-XhoI fragment containing hygromycinB resistant gene was cloned to prepare plasmid #5C.

Example 4 Preparation of DNA Fragments Carrying Ten Exogenous Genes (4)(see FIG. 9)

A total of 500 ng including 100 ng of a DNA fragment containing fireflyluciferase gene and Renilla luciferase gene cut out from plasmid #1Cwith SapI, 100 ng of a DNA fragment containing EGFP gene and puromycinresistant gene cut out from plasmid #2C with SapI, 100 ng of a DNAfragment containing Cypridina noctiluca luciferase gene and E2-Crimsongene cut out from plasmid #3C with SapI, 100 ng of a DNA fragmentcontaining EBFP2 gene and Zeocin resistant gene cut out from plasmid #4Cwith SapI, and 100 ng of a DNA fragment containing dKeima-Red gene andhygromycin B resistant gene cut out from plasmid #5C with SapI wasdissolved in 5 μL, of H₂O, and the solution was mixed with 5 μL ofLigation-Convenience Kit (NIPPON GENE Co., Ltd.) and allowed to react at16° C. for 60 minutes. After purification, the product was dissolved in7 μL of H₂O, and 1 μL of plasmid #6 (pDONR-221, Life Technologies, Inc.)(150 ng) and 2 μL of BP Clonase2 (Life Technologies, Inc.) were addedand allowed to react at 25° C. for 2 hours, and then the product wasintroduced into Escherichia coli DH-5α, and a kanamycin resistant colonywas isolated to prepare plasmid #7.

Example 5 Preparation of Template DNA for Forming Stealth RNA CarryingTen Exogenous Genes (see FIG. 10)

Plasmid #8 is prepared by replacing the kanamycin resistant gene ofplasmid pACYC177 having a replication origin of p15A (Non-PatentDocument 56) with a DNA fragment containing attB1-chloramphenicolresistant gene-attB2 of pDONR-221. The DNA fragment in which attB1, T7terminator, and HDV ribozyme are connected in sequence on 5′ side of aDNA, containing hN-hC-hPolS optimized with OptimumGen Gene Design System(SEQ ID NO: 53) was synthesized by GenScript. Similarly, the DNA inwhich T7 promoter and attB2 are connected on 3′ side of a DNA containinghPolL optimized with OptimumGen Gene Design System (SEQ ID NO: 54) wassynthesized. A total of 300 ng including 100 ng of a DNA fragmentcontaining attB1-T7 terminator-HDV ribozyme-hN-hC-hPolS in this ordercut out with BamHI and XmaI, 100 ng of a DNA fragment containing tengenes cut out from plasmid #7 with XmaI and NotI, and 100 ng of a DNAfragment containing hPolL-T7 promoter-attB2 in this order cut out withNotI and SalI was dissolved in 5 μL of H₂O, and the solution was mixedwith 5 μL of Ligation-Convenience Kit and allowed to react at 16° C. for60 minutes. After purification, the product was dissolved in 7 μL ofH₂O, and 1 μL of plasmid #8 (150 ng) and 2 μL of BP Clonase2 were addedand allowed to react at 25° C. for 16 hours, and then the product wasintroduced into Escherichia coli HST-08 (Takara Bio Co.), and anampicillin resistant colony was isolated to prepare plasmid #9B which isto be a template for synthesizing a negative-sense stealth RNA.

A template DNA for synthesizing a positive-sense stealth RNA is preparedby replacing T7 promoter with T7 terminator. Specifically, a total of300 ng including 100 ng of a DNA fragment containing attB1-T7promoter-hN-hC-hPolS in this order, 100 ng of a DNA fragment containingten gene cut out from plasmid #7 with XmaI and NotI, and 100 ng of a DNAfragment containing hPolL-HDV ribozyme-T7 terminator-attB2 in this ordercut out with NotI and SalI was dissolved in 5 μL in H₂O, and thesolution was mixed with 5 μL of Ligation-Convenience Kit and allowed toreact at 16° C. for 60 minutes. After purification, the product wasdissolved in 7 μL of H₂O, and 1 μL of plasmid #8 (150 ng) and 2 μL of BPClonase2 were added and allowed to react at 25° C. for 16 hours, andthen the product was introduced into Escherichia coli HST-08, and anampicillin resistant colony was isolated to prepare plasmid #9A which isto be a template for synthesizing a positive-sense stealth RNA.

Example 6 Reconstitution of Stealth RNA Gene Expression System CarryingTen Exogenous Genes (Method 1) (see FIG. 11)

Method 1 was conducted according to the method described in PatentDocument 3 and Non-Patent Document 7.

Specifically, as BHK/T7/151M (SE) cells, BHK-21 cells derived fromhamster in which T7 RNA polymerase and M protein are stably expressedwere prepared in the following manner. BHK-21 cells were obtained fromRIKEN BioResource Center. A cDNA synthesized by optimizing codons of T7RNA polymerase gene (Non-Patent Document 74) for animal cells (Sequenceinformation 77) was installed on a retrovirus vector pCX4neo (Non-PatentDocument 75, GenBank Accession Number AB086385) and introduced intoBHK-21 cells, and then selected in 10% FCS-containinq DMEM culturemedium containing 800 μg/mL of G-418 was conducted, and BHK/T7 cellswere obtained. Next, M gene of Sendai virus temperature-sensitive mutantClone 151 strain (GenBank Accession Number NM_011046) was installed on aretrovirus vector pCX4pur (Non-Patent Document 75, GenBank AccessionNumber AB086386), and introduced into BHK-21/T7 cells, and thenselection in 10% FCS-containing DMEM culture medium containing 200μg/min of Puromycin was conducted, and thus BHK/T7/151M (SE) cells wereobtained.

Expression vectors used in reconstitution were prepared in the followingmanner. A plasmid pCMV-NP for expressing NP protein, a plasmid pCMV-Pfor expressing P protein, an plasmid pCMV-L for expressing L protein,and plasmid pCMV-Furin for expressing mouse Furin were prepared byrespectively connecting NP gene, P gene, and L gene (GenBank AccessionNumber M30202.1) of Sendai virus Z strain, and mouse Furin cDNA(Non-Patent Document 76, GenBank Accession Number NM_011046) downstreamthe enhancer and the promoter of the Immediate Early gene ofCytomegalovirus (Non-Patent Document 77). A plasmid pSRD-HN-Fmut(Non-Patent Document 78) for expressing F and HN proteins is a plasmidin which F and HN genes of Sendai virus Z strain are connecteddownstream the SRα promoter (Non-Patent Document 79). pMKIT-151M wasprepared by connecting M gene of Sendai virus temperature-sensitivemutant Clone 151 strain downstream the SRα promoter.

BHK/T7/151M (SE) cells stably expressing M protein were seeded on a6-well plate at 5×10⁵ cells/well, and cultured for 24 hours, and thenwashed. Plasmid #9A, a plasmid pCMV-NP for expressing NP protein, aplasmid pCMV-P for expressing P protein, a plasmid pCMV-L for expressingL protein, a plasmid pSRD-HN-Fmut for expressing F and HN protein, and aplasmid pCMV-Furin for expressing mouse Furin were suspended in 300 μLof OptiMEM (Life Technologies, Inc.) in a quantitative ratio of 2 μg, 1μg, 1 μg, 1 μg, 2 μg, and 20 ng, respectively, and the suspension wasmixed with 300 μL of OptiMEM containing 10 μL of Lipofectamine LTX (LifeTechnologies, Inc.) and left at room temperature for 20 minutes. Theculture medium thus prepared was added to cells and the cells werecultured for 4 hours. After washing the cells again, a 10%FCS-containing DMEM culture medium was further added, and the cells werefurther cultured at 32° C. for 3 days. Then the cells were transferredto a 10% FCS-containing DMEM culture medium containing 300 μg/mL ofhygromycin B, and cultivation was continued, and BHK/#9A cells wereseparated. Occurrence of the reconstitution of the stealth RNA geneexpression system was confirmed by the expression of EGFP and Keima-Red.

Example 7 Reconstitution of Stealth RNA Gene Expression System CarryingTen Exogenous Genes (Method 2) (see FIG. 12)

Escherichia coli E-AIST7 strain which is a double deletion mutant ofRecA and RNaseE was prepared by disrupting RNase E gene and RecA geneEscherichia coli BL21 (DE3) strain (Non-Patent Document 68) in thisorder. Deletion mutation of C-terminus of RNase E (rne131) wasintroduced into RNase E gene (Non-Patent Document 59), and completedeletion mutation was introduced into Rec A gene. The gene disruptionwas conducted by using Quick & Easy E. coli Gene Deletion Kit availablefrom Gene Bridges GmbH according to the protocol of the kit. Plasmid #10for expressing single-stranded RNA binding protein (N) in Escherichiacoli was prepared by carrying N gene having codons optimized forEscherichia coli (eN) (SEQ ID No: 55) on plasmid pET-24a (+) (MerckKGaA).

Plasmid #9B and plasmid #10 were introduced into Escherichia coliE-AIST7 strain, and an E-AIST7/N/9B strain was prepared by selectionwith ampicillin and kanamycin. E-AIST7/N/9B strain was cultured at 30°C., and at OD₆₀₀=0.3, 0.5 mM IPTG was added to induce expression of T7RNA polymerase, and cultured for 3 hours and Escherichia coli wascollected. The collected cells were suspended in 10 mL of 10% Sucrose,50 mM Tris-HCl (pH 7.5), 2 mM MgCl₂, and after addition of 150 k unitsof rLysazome (Merck KGaA) and 25 units of Benzonase (Merck KGaA), thecells were treated at 30° C. for 30 minutes, and protoplasts werecollected. The protoplasts were broken with 50 mM Tris-HCl (pH 7.5), 2mM MgCl₂, 50 mM CHAPS, and a supernatant from centrifugation at 4,500rpm for 10 minutes was centrifuged at 25,000 rpm for 60 minutes with aBeckman SW41Ti rotor, and an RNA-N protein complex was collected as aprecipitate. The RNA-N protein complex was further suspended in 28%calcium chloride, and centrifuged at 37,000 rpm for 45 hours with aBeckman SW41Ti rotor, and thus the RNA-N protein complex was purified.

BHK/T7/151M (SE) cells were seeded on a 6-well plate at 5×10⁵cells/well, and cultured for 24 hours, and then each 1 μL of plasmidpCMV-P for expressing P protein, and plasmid pCMV-L for expressing Lprotein were introduced by Lipofectamine LTX. After another 24 hours, 5of the RNA-N protein complex was mixed with 10 μL of a Pro-DeliverINreagent (OZ Biosciences) and introduced into the cells. From after 24hours, the cells were transferred to a 10% FCS-containing DMEM culturemedium containing 300 μg/mL of hygromycin B and cultivation wascontinued, and BHK/#9A2 cells were separated. Occurrence ofreconstitution of the stealth RNA gene expression system was confirmedby the expression of EGFP and Keima-Red.

Example 8 Preparation of Stealth RNA Vector #1 Carrying Ten ExogenousGenes

For 5.0×10⁵ BHK/#9A cells (or BHK/#9A2 cells), pMKIT-115M, pSRD-HN-Fmut,and pCMV-Furin which are defective gene expression plasmids wereintroduced ratio of 2 μg, 2 μg, and 30 ng with Lipofectamine LTX, andafter washing the cells after 4 hours, a 10% FCS-containing DMEM culturemedium was added, and the cells were further cultured at 32° C. for 4days. Then the culture supernatant containing stealth RNA vector #1(FIG. 13) was collected, and filtered through a 0.45 μm filter, and thevector was concentrated by ultracentrifugation as necessary. The vectorsuspension as rapidly frozen with liquid nitrogen, and stored at −80° C.The activity of the vector was assayed by an indirect fluorescentantibody method by an anti-NP protein ant body by using LLCMK₂ cellsderived from monkey kidney (Non-Patent Document 7). The infectivitytiter of the stealth RNA vector obtained by the present method was about10⁷ infectious units/mL, and equivalent or higher activity was obtainedas compared with a conventional persistent expression type Sendai virusvector.

Example 9 Gene Expression by Stealth RNA Vector Carrying Ten ExogenousGenes (see FIG. 14)

HeLa cells were infected with stealth RNA vector #1 prepared in (Example8) at MOI=3, and HeLa/#9 cells were established by selection in a 10%FCS-containing DMEM culture medium containing 100 μg/mL of hygromycin B.The drug resistance of these cells was selected by puromycin (1.5μg/mL), Zeocin (100 μg/mL), hygromycin B (100 μg/mL), G418 (800 μg/mL),and Blasticidin S (10 μg/mL), and the survival rate was measured by acolony assay. It was confirmed HeLa/#9 cells showed resistanceselectively to puromycin, Zeocin, and hygromycin B and expressed theresistance characters to these three drugs unlike the negative controlHeLa cells that are sensitive to all of these antibiotics (FIG. 14,upper stage).

Expression of fluorescent proteins in HeLa/#9 cells was measured with aflow cytometer (Gallios, Beckman Coulter). Observation conditions ofindividual fluorescent proteins are as follows. EBFP2: excitation 405nm, detection 450 nm; Keima-Red: excitation 405 nm, detection 620 nm;EGFP: excitation 488 nm, detection 530 nm; E2-Crimson: excitation 638nm, detection 660 nm. It was confirmed that HeLa/#9 cells significantlyexpress four fluorescent proteins as compared with HeLa cells not havinga vector (FIG. 14, middle stage).

Expression of luciferase in HeLa/#9 cells was examined by detecting theemission with a luminometer (Promega, Corp.) by using the followingreagents. Firefly luciferase and Renilla luciferase: Dual-LuciferaseReporter Assay System (Promega, Corp.); Cypridina noctiluca luciferase:BioLux Cypridina Luciferase Assay Kit (New England Biolabs, Inc.).Activity of any luciferase was not detected in HeLa/#9 cells not havinga vector, but high activity was detected in HeLa/#9 cells (FIG. 14,lower stage).

Example 10 Preparation of Stealth RNA Vector #2 Carrying Ten ExogenousGenes (FIG. 15)

A vector was prepared in the same manner as described in (Example 8) andverified in the manner as described in (Example 9) except that hN, hC,hPolS, hPolL genes used for preparation of the template cDNA wereoptimized by GeneGPS Expression Optimization Technology, and threegenes, hN, hC, and hPolS were installed in the order of hN-hPolS-hC. TheDNA fragment in which attB1 and T7 promoter are connected in this orderon 5′ side of the DNA containing hN-hPolS-hC (SEQ ID NO: 78) wassynthesized by DNA 2.0. Similarly, the DNA fragment in which HDVribozyme, T7 terminator, and attB2 are connected on 3′ side of the DNAcontaining hPolL (SEQ ID NO: 79) was synthesized by DNA 2.0.

Example 11 Preparation of Stealth RNA Vectors #3 and #4 (FIG. 16)Carrying Ten Exogenous Genes

A vector was prepared in the same manner as described in (Example 8) andverified in the manner as described in (Example 9) except that threegenes, hN, hC, and hPolS optimized by OptimumGen Gene Design System wasinstalled in the order of hN-hPolS-hC (#3) or in the order ofhPolS-hN-hC (#4). The DNA fragment in which attB1 and T7 promoter areconnected in this order on 5′ side of the DNA containing hN-hPolS-hC(SEQ ID NO: 80), the DNA fragment in which attB1 and T7 promoter areconnected in this order on 5′ side of the DNA containing hPolS-hN-hC(SEQ ID NO: 81), and the DNA fragment in which HDV ribozyme, T7terminator, and attB2 are connected on 3′ side of the DNA containinghPolL (SEQ ID NO: 82) were synthesized by GenScript.

Example 12 Preparation of Stealth RNA Vector #5 Carrying Four ExogenousGenes

Blasticidin S resistant gene (Non-Patent Document 69) (SEQ ID NO: 56)and Kusabira-Orange gene (Non-Patent Document 70) (GenBank AccessionNumber AB128819) were amplified to have a structure of Acc65I-cDNA-XhoIPCR, and sub-cloned (FIG. 7). Plasmid #5D is obtained by cloning the DNAhaving the following structure between the ApaI cleavage site and theStuI cleavage site of LITMUS38i. However, the SapI digested end isdifferent from that of plasmid #5: SapI cleavage site-SEQ ID NO: 1-SEQID NO: 36-Acc65I cleavage site-SalI cleavage site-SEQ ID NO: 37-SEQ IDNO: 4-ctt-SEQ ID NO: 1-SEQ ID NO: 38-BsiWI cleavage site-XhoI cleavagesite-SEQ ID NO: 39-SEQ ID NO: 4-attB2-SapI cleavage site.

Between Acc65I-SalI plasmid #1, an Acc65I-XhoI fragment containingdKeima-Red gene was cloned to prepare plasmid #1D. Further, betweenBsiWI-XhoI of plasmid #1D, an Acc65I-XhoI fragment containingBlasticidin S resistant gene was cloned to prepare plasmid #1E. Further,between Acc65I-SalI of plasmid #5D, an Acc65I-XhoI fragment containingEGFP gene was cloned to prepare plasmid #5E. Further, between BsiWI-XhoIplasmid #5E, an Acc65I-XhoI fragment containing Kusabira-Orange gene wascloned to prepare plasmid #5F.

A total of 200 ng including 100 ng of a DNA fragment containingdKeima-Red gene and Blasticidin S resistant gene cut out from plasmid#1E with SapI, and 100 ng of a DNA fragment containing EGFP gene andKusabira-Orange gene cut out from plasmid #5F with SapI was dissolved in5 μL of H₂O, and the solution was mixed with 5 μL ofLigation-Convenience Kit and allowed to react at 16° C. for 60 minutes.After purification, the product was dissolved in 7 μL of H₂O, and 1 μLof plasmid #6 (150 ng) and 2 μL of BP Clonase2 were added and allowed toreact at 25° C. for 2 hours, and then the product was introduced intoEscherichia coli DH-5α, and a kanamycin resistant colony was isolated toprepare plasmid #11. Preparation of stealth RNA vector #5 using the DNAfragment containing four genes cut out from plasmid #11 with XmaI andNotI was conducted in the manner as described in (Example 5) to (Example8).

Example 13 Induction of IFN-β Gene by Stealth RNA Vector (FIG. 17)

Defective and persistent Sendai virus vector SeVdp (KR/Bsr/EGFP/KO) isdescribed in Non-Patent Document 7. Primary culture human skin-derivedfibroblasts were infected with stealth RNA vector #5 prepared in(Example 12), and SeVdp (KR/Bsr/EGFP/KO) vector at MOI=3 each. Underthis condition, both of the vectors could introduce genes into about 80%of the cells. At 24 hours after infection with the vectors, total RNA ofcells was extracted by using ISOGEN Kit (NIPPON GENE Co., Ltd.), andgenomic DNA was degraded by using Deoxyribonuclease (RT Grade) (NIPPONGENE Co., Ltd.). Next, using this RNA as a template, First strand cDNAsynthesis was conducted by reverse transcription reaction by usingSuperScriptIII First-Strand Synthesis system for RT-PCR (LifeTechnologies, Inc.) and oligo(dT) 20. Further, by using SsoAdvancedUniversal SYBR Green Supermix (Bio-Rad), and using the first strand cDNAas a template, an expression amount of IFN-β mRNA was analyzed by thereal-time PCR method using Gene Specific Primers (GSP) of a referencegene or interferon beta gene, and CFX96 Real-Time System (Bio-Rad).

Example 14 Preparation of Stealth RNA Gene Expression Systems #6, #7,#8, 9 and #10 Carrying Six Exogenous Genes (FIG. 70 and FIG. 22)

Plasmid #2D was obtained by cloning the DNA having the followingstructure between the ApaI cleavage site and the StuI cleavage site ofplasmid LITMUS38i. However, the SapI digested end is different from thatof plasmid #2: SapI cleavage site-SEQ ID NO: 1-SEQ ID NO: 28-Acc65Icleavage site-SalI cleavage site-SEQ ID NO: 29-SEQ ID NO: 4-ctt-SEQ IDNO:1-SEQ ID NO: 30-BsiWI cleavage site-XhoI cleavage site-SEQ ID NO:31-SEQ ID NO: 4-SapI cleavage site.

Between Acc65I-SalI plasmid #2D, an Acc65I-XhoI fragment containing EGFPgene was cloned to prepare plasmid #2E. Further, between BsiWI-XhoI ofplasmid #2E, an Acc65I-XhoI fragment containing puromycin resistant genewas cloned to prepare plasmid #2F.

A total of 300 ng including 100 ng of a DNA fragment containing fireflyluciferase gene and Renilla luciferase gene cut out from plasmid #1Cwith SapI, 100 ng of a DNA fragment containing EGFP gene and puromycinresistant gene cut out from plasmid #2F with SapI, and 100 ng of a DNAfragment containing dKeima-Red gene and hygromycin Bresistant gene cutout from plasmid #5C with SapI was dissolved in 5 μL of H₂O, and thesolution was mixed with 5 μL of Ligation-Convenience Kit and allowed toreact at 16° C. for 60 minutes. After purification, the product wasdissolved in 7 μL of H₂O, and 1 μL of plasmid #6 (150 ng) and 2 μL of BPClonase2 were added and allowed to react at 25° C. for 2 hours, and thenthe product was introduced into Escherichia coli DH-5α, and a kanamycinresistant colony was isolated to prepare plasmid #12. Preparation ofstealth RNA gene expression systems #6, #7 and #8 (FIG. 20) (Example 16)and #9 and #10 (FIG. 22) (Example 18) using the DNA fragment containingsix genes cut out from plasmid #12 with XmaI and NotI was conducted inthe manner as described in (Example 5), (Example 6) and (Example 8).

Example 15 Preparation of Stealth RNA Gene Expression Systems #11, #12,#13, #14 and #15 Carrying Five Exogenous Genes (FIG. 18)

Plasmid #13 carrying five genes was prepared in the manner as describedin (Example 14) except that among the genes installed plasmid #12 in(Example 14), firefly luciferase gene was deleted, and puromycinresistant gene was replace by tetracycline resistant gene derived fromplasmid pBR322 (GenBank Accession Number J01749.1).

These five exogenous genes were installed on stealth RNA vector #3 (FIG.16) carrying three genes, hN, hC, and hPolS in the order of hN-hPolS-hC,and stealth RNA gene expression system #11 carrying five exogenous geneswere prepared. Further, in the XmaI site of this stealth RNA geneexpression system, a gene cassette containing codon-optimized RIG-IC(SEQ ID NO: 83), a gene cassette containing codon-optimized C-terminalregion of Sendai virus protein (SEQ ID NO: 84), or a gene cassettecontaining codon-optimized PSMA7 which is a constituent of proteasome(SEQ ID NO: 85) was inserted, and thus stealth RNA gene expressionsystems #12, #13 and #14 carrying five exogenous genes were prepared.Stealth RNA gene expression system #15 carrying five exogenous genes isdesigned to express V protein by replacing part of hPolS gene ofhN-hPolS-hC gene with P gene of non-optimized Sendai virus Z strain (SEQID NO: 86).

From cells containing these stealth RNA gene expression systems, stealthRNA vectors were prepared according to Example 8, and introduced intohuman-derived 293 cells, and interferon inducibility was measured. InFIG. 19, stealth RNA vectors #11 and #12 were compared asrepresentative, and it was shown that by adding RIG-IC gene, inductionof interferon beta remaining in the stealth RNA vector is almostcompletely suppressed.

Example 16 Analysis of Influence of Variation in Expression Efficiencyof N Protein and C Protein on Expression of Exogenous Genes Installed onStealth RNA Gene Expression System (see FIG. 20)

In front of the translation initiation codon (AUG) of firefly luciferasecDNA encoded by pGL4.12 (Promega Corporation) (GenBank Accession NumberAY738224), an RNA sequence corresponding to SEQ ID NO: 55, SEQ ID NO: 57or SEQ ID NO: 58 was inserted, and firefly luciferase was expressed inHeLa cells by using a CMV promoter, and activity of luciferase wasexamined by using Dual-Luciferase Reporter Assay System (FIG. 20). Itwas demonstrated that when an another initiation codon is placed atout-frame position and upstream to the authentic translation initiationcodon, the translating frame is shifted from the original translatingframe of the protein, and the translation efficiency is deteriorated.

Next, stealth RNA gene expression systems #6, #7 and #8 in which a basesequence on 5′ upstream side of the translation initiation codon of hNmRNA and hC mRNA was modified were examined for their gene expressionability. In stealth RNA gene expression system #6, a so-called “Kozaksequence (SEQ ID NO: 57)” which is be to provide the highest translationefficiency is positioned on 5′ upstream side of the translationinitiation codon (AUG) of hN mRNA and hC mRNA. In stealth RNA geneexpression system #7, 5′ upstream side of the translation initiationcodon of hN mRNA is replaced by “Kozak sequence (SEQ ID NO: 57)”(Non-Patent Document 60) and 5′ upstream side of the translationinitiation codon of hC mRNA is replaced by the base sequence of SEQ IDNO: 59 that lowers the translation efficiency to 23%. In stealth RNAgene expression system #8, 5′ upstream side of the translationinitiation codon of hN mRNA is replaced by the base sequence of SEQ IDNO: 58 that lowers the translation efficiency to 40%, and 5′ upstreamside of the translation initiation codon of hC mRNA is replaced by thebase sequence of SEQ ID NO: 59 that lowers the translation efficiency to23%. In this experiment, activity of luciferase was examined inBHK/T7/151M (SE) cells that stably retain stealth RNA gene expressionsystems #6, #7 or #8, by using Dual-Luciferase Reporter Assay System(FIG. 20).

Example 17 Preparation of Stealth RNA Gene Expression Systems #16 and#17 Carrying Five Exogenous Genes (FIG. 21)

Stealth RNA gene expression system #11 in which three genes, hN, hC, andhPolS are installed in the order of hN-hPolS-hC, and five exogenousgenes are installed has been described in (Example 15). In hC gene ofthis vector, the translation efficiency is lowered to 23% bymodification of the sequence of 5′ untranslated region (FIG. 21).Stealth RNA gene expression system #16 is a system in which hC gene isremoved from #11. The stealth RNA gene expression system #17 is a systemin which the 5′ untranslated sequence of hC gene in #11 is replaced bythe Kozak sequence to achieve 100% of the translation efficiency.

As can be realized from the expression of EGFP shown in FIG. 21, thegene expression level of the stealth RNA gene expression system can beregulated by changing the translation efficiency of hC gene. Although hCgene is not an essential element for reconstitution of a stealth RNAgene expression system, lack of hC gene results in very strongexpression of exogenous genes, and thus proliferation of cells issuppressed. Therefore, it is realistic to obtain gene expression of thepractical level by allowing a certain degree of expression of hC gene.

Example 18 Analysis of Influence of Packaging Signal on Genome 3′ Sideof Stealth RNA Gene Expression System on Production of Vector Particle(see FIG. 22)

Stealth RNA gene expression system #9 is a system in which sequence D on3′ side of genome RNA (SEQ ID NO: 17) is deleted from stealth RNA geneexpression system #6 (FIG. 20). Stealth RNA gene expression system #10is a system in which sequence D on 3′ side of genome RNA (SEQ ID NO: 17)is deleted from stealth RNA gene expression system #7 (FIG. 20). InBHK/T7/151M (SE) cells stably retaining these stealth RNA geneexpression systems, proteins M, F, and HN were expressed in the manneras described in (Example 5), and the gene introduction ability of thestealth RNA vector collected in the supernatant was assayed by anindirect fluorescent antibody method using an anti-NP protein antibodyand LLCMK₂ cells (Non-Patent Document 7).

Further, this sequence of 18 nucleotides was replaced by an arbitrarilyselected partial sequence of mRNA derived from House-keeping generecited in (Table 1) ((5) of FIG. 2 (SEQ ID NO: 75)), and no variationwas observed in the particle formation efficiency (data not shown).

From the above, it can be considered that the region having a length of18 nucleotides from the 97th to 114th nucleotides from 3′ terminus ofthe genome or a region having a partial length thereof is an essentialfor packaging for particle formation in the negative-sensesingle-stranded RNA. In any case, it can be concluded that the regionhaving a length of 18 nucleotides or a region having a partial lengththereof at this position is “packaging signal region” that is essentialfor the stealth RNA gene expression system to be incorporated into thevirus-like particle, although it is not essential for transcription andreplication from the negative-sense single-stranded RNA as a template.

Example 19 Variation with Time of Luciferase Activity When HeLa CellsRetaining Stealth RNA Gene Expression System are Treated with siRNA (seeFIG. 23)

Stealth RNA vector #6 was prepared from stealth RNA gene expressionsystem #6 (FIG. 20), and gene introduction into HeLa cells and selectionwith hygromycin B were conducted, and thus HeLa/#3 cell strain wasestablished. HeLa/#3 cells were seeded on a 48-well plate at1.0×10⁴/well, and on the next day, siRNA targeting a target sequence ofPolL gene (SEQ ID NO: 46) was mixed with an introducing reagent RNAiMAX(Life Technologies, Inc.) in a final concentration 100 nM and introducedinto the cells. Luciferase activity was measured over time, and in allthe four independent experiments, luciferase activity was suppressed toabout 0.1% in 10 days. This reveals taht the stealth RNA gene expressionsystem was removed from cells efficiently.

Example 20 Preparation of Stealth RNA Vector Carrying Giant Gene (seeFIG. 24)

Stealth RNA vectors carrying various exogenous genes can be prepared byproducing “transcription cassettes” each consisting of two genes in thesame manner as in (Example 1) to (Example 2), sequentially linking the“transcription cassettes” in the same manner as in (Example 3) to(Example 5), and preparing in the same manner as in (Example 6) or(Example 7) and (Example 8). Names and base sequences of exogenous genesthat can be installed as such a giant exogenous gene are as follows.Human KLF4: SEQ ID NO: 60, human OCT4: SEQ ID NO: 61, human SOX2: SEQ IDNO: 62, human c-Myc: SEQ ID NO: 63, human BRG1: SEQ ID NO: 64, humanBAF155: SEQ ID NO: 65, human immunoglobulin G H chain: SEQ ID NO: 66,human immunoglobulin G L chain: SEQ ID NO: 67, human immunoglobulin Mclone: 2G9 H chain: SEQ ID NO: 68, human immunoglobulin M clone 2G9 Lchain: SEQ ID NO: 69, human immunoglobulin M J chain: SEQ ID NO: 70,human blood coagulation factor VIII: SEQ ID NO: 71, and humandystrophin: SEQ ID NO: 72.

RNA expression systems carrying these genes as exogenous genes can beintroduced into target cells in the technique corresponding to theprocedure described in the foregoing Examples. By expressing pluralexogenous genes simultaneously in the same cell, it becomes possible toadd a desired modification such as cell-reprogramming to the introducedcells.

Example 21 Induction of Induced Pluripotent Stem Cells (iPS Cells) byStealth RNA Vector Carrying Six Reprogramming Genes (FIG. 25)

The capability of carrying six or more genes and expressing themsecurely, which is a feature of the stealth RNA vector would beparticularly effective for cell-reprogramming in which human somaticcells are initialized and converted to iPS cells. Thus, the presentinventors prepared a stealth RNA vector simultaneously expressing atotal of six reprogramming genes by adding reprogramming genes NANOG andLIN28 (Patent Document 2, and Non-Patent Document 2) havingcomplementary functions to the combination of four reprogramming genes,KLF4, OCT4, SOX2, and c-MYC that was first reported as a method formaking human induced pluripotent stem cells (Patent Document 1, andNon-Patent Document 1), and compared the cell-reprogramming activitybetween the stealth RNA, vector and the “persistent expression typeSendai virus vector simultaneously carrying the four reprogramming genes(KLF4, OCT4, SOX2 and c-MYC)” having the highest reprogrammingefficiency among the iPS cell preparation techniques that have beenreported heretofore (Patent Document 3, Patent Document 4, andNon-Patent Document 7) (FIG. 25A).

Stealth RNA vector #23 carrying six reprogramming genes (FIG. 25B) wasprepared according to Example 6 and Example 8 by binding human KLF4 (SE0ID NO: 60), human OCT4 (SEQ ID NO: 61), human SOX2 (SEQ ID NO: 62),human c-MYC (SEQ ID NO: 63), human NANOG (SEQ ID NO: 87), and humanLIN28 (SEQ ID NO: 88) in this order by the method shown in Example 14,and incorporating the genes into stealth RNA vector #3 of FIG. 16.

Preparation of iPS cells was conducted according to Patent Document 3.To be more specific, TIG3 cells derived from human embryonic fibroblastswere seeded on a 12-well plate at 1.0×10⁵ cells/well, and on the nextday, a Sendai virus vector for persistent expression carrying KLF4,OCT4, SOX2, and c-MYC (FIG. 25A), and a stealth RNA vector carryingKLF4, OCT4, SOX2, c-MYC, NANOG, and LIN28 (FIG. 25B) were added into theculture medium in the condition of MOI (Multiplicity of Infection)=3,and left still for 2 hour at room temperature, and then culturedovernight at 37° C. to infect the cells. MEF treated with mitomycin Cwas prepared as feeder cells on a gelatin-coated dish, and theaforementioned cells transfected with the vector were seeded thereon,and cultured in a culture medium for human multipotent stem cellsStemFit AK03 (Ajinomoto, Co., Inc.). At 11 days after gene introduction,cells were stained with AlexaFluor488-labeled anti-TRA-1-60 antigenantibody (Merck-Millipore), and the number of clones of TRA-1-60positive iPS cells appeared from 1×10⁴ TIG-3 cells were counted (FIG.25C). While 85 clones of iPS cell clones appeared by thefour-factor-carrying vector (reprogramming efficiency 0.35%), 4290clones of iPS cell clones appeared by the six-factor-carrying vector(reprogramming efficiency 42.9%), revealing the effectiveness of thestealth RNA vector carrying six genes. The total nucleotide length ofgenes used in the present Example is 7.0 kb, and this size cannot berealized by method using a conventional RNA vector.

Example 22 Production of Human Immunoglobulin M by SimultaneousExpression of H chain, L chain, and J chain of Human Immunoglobulin M(IgM) (FIG. 26)

As representative product for which simultaneous expression of pluralpolypeptides is required in the field of production ofbiopharmaceuticals, antibody drugs are recited. While the commercialproduction technology of immunoglobulin G (IgG) capable of expressingand producing H chain and L chain has been already established,production of IgM for which simultaneous expression of three genesencoding H chain, L chain, and J chain are required is not still easytoday (Non-Patent Document 84). It is known that in IgM, there is anantibody having strong antitumor activity that is not present in IgG(Non-Patent Document 85), and establishment of a production method ofIgM is industrially very significant. Thus, the present inventorsattempted to produce an IgM having a molecular weight of 950 k Dalton bcarrying three genes that encode H chain, L chain and J chain of humanIgM on a stealth RNA vector and expressing them simultaneously.

In Example 22, human monoclonal IgM antibodies 9F11 and 2G9 that reactwith the cells infected with human immunodeficiency virus (HIV)(Non-Patent Document 86) were selected as a material and a set of Hchain gene (SEQ ID NO: 89) and L chain gene (SEQ ID NO: 90) of 9F11antibody, J chain gene (SEQ ID NO: 70), and hygromycin B resistant gene(SEC ID NO: 50), or a set of H chain gene (SEQ ID NO: 68) and L chaingene (SEQ ID NO: 69) of 2G9 antibody, J chain gene (SEQ ID NO: 70), andhygromycin B resistant gene (SEQ ID NO: 50) were linked in this orderaccording to Example 12, and installed on stealth RNA vector #8 of FIG.20, to obtain stealth RNA vectors #23 and #20. Then gene introductioninto BHK cells derived from hamster acclimated to a serum-free culturemedium, Opti-Pro SFM (Lite Technologies, Inc.) for protein productionwas conducted in the condition of MOI=3, and selection was conducted byadding 100 μg/mL hygromycin B. After renewing the culture medium, cellswere collected after 24 hours of culture, and the culture supernatantwas collected.

The amount of human IgM in the culture supernatant was quantified by ananti-human IgM ELISA kit (Bethyl Laboratories, Inc.), and 9.17 μg/mL ofIgM was detected when the gene set of 2G9 was introduced, and 11.15μg/mL of IgM was detected when the gene set of 9F11 was introduced. IgMin the culture supernatant of BHK cells into which genes were notintroduced was under or equal to the detection limit. Expressionefficiency per cell per day (pg/cell/day) converted from the aboveamount was 16.38 pg/cell/day for 2G9, and 19.91 pg/cell/day for 9E11(FIG. 26).

Then, the culture supernatants containing 300 ng and 100 ng of IgM wereanalyzed by SDS polyacrylamide gel electrophoresis using 4-20% GradientGel (Bio-Rad), and stained with BioSafe Coomasie G250 stain (Bio-Rad).Under a non-reduced condition, a band was detected at the position of970 kDa as is the same with native human IgM, and under a reducedcondition, bands were detected at the positions of H chain of molecularweight of 75 kDa and L chain of 25 kDa. This reveals that an IgMmolecule in which 21 polypeptides are bound, that is the same with thenative one is generated.

In Non-Patent Document 84, analytical results in four clones of cellsstably expressing IgM obtained as a result of gene amplification withmethotrexate by using CHO-DG44 cells and HEK293 cells are described, andthe expression efficiency was 25.00, 3.59, 4.60, and 0.21 pg/cell/day,respectively. This reveals that by using a stealth RNA vector, it ispossible to easily realize production at an equivalent or higher levelcompared with expression of IgM achieved by gene amplification thatrequires several months.

Example 23 Production of Human Bispecific Antibody by SimultaneousExpression of Four cDNAs (FIG. 27)

Recently, bispecific antibodies capable of recognizing two differentantigens attract attentions in the field of biopharmaceuticals as amolecule that greatly extends the possibility of the existing antibodydrugs. A bispecific antibody is a tetramer made up of H chain (A) and Lchain (A) that recognize antigen A, and H chain (B) and L chain (B) thatrecognize antigen B, and is prepared by introducing a mutation so that Hchain (A) and L chain (B), and H chain (B) and L chain (A) are difficultto bind each other, and introducing a mutation so that binding between Hchain (A) and H chain (B) is stronger than binding between H chains (A)or binding between H chains (B), and then expressing four genes encodingH chain (A), L chain (A), H chain (B), and L chain (B) simultaneously(Non-Patent Document 87). Since it is very difficult to obtain a cellstrain that simultaneously expresses four polypeptides by geneamplification after simultaneous introduction of these four genes intocells, it is normally produced by transient gene expression. In thepresent Example, the present inventors attempted to prepare HEDesignLKthat simultaneously recognizes HER2 and an epithelial growth factorreceptor (EGFR) among the bispecific antibodies described in Non-PatentDocument 87.

H chain HC1(VH_(VRD1)CH1_(CRD2)) gene (SEQ ID NO: 91) and L chain LC1(VL_(VRD1)Cλ_(CRD2)) gene (SEQ ID NO: 92) of anti-HER2 antibody, and Hchain HC2 (VH_(VRD2)CH2_(WT)) gene (SEQ ID NO: 93) and L chain LC2(VL_(VRD2)Ck_(WT)) gene (SEQ ID NO: 94) of anti-EGFR antibody disclosedin Non-Patent Document 87 were linked together with EGFP gene andhygromycin B resistant gene according to Example 14, and installed onstealth RNA vector #8 (FIG. 20) to prepare stealth RNA vector #24. Forcomparison, vector #25 for expressing only H chain and L chain ofanti-HER2 antibody (FIG. 27B) and vector #26 for expressing only H chainand L chain of anti-EGFR antibody (FIG. 27C) were prepared according toExample 12.

Using these vectors, genes were introduced into BHK cells derived fromhamster acclimated to Opti-Pro SFM (Life Technologies, Inc.) by themethod of Example 22, and an amount of human IgG in the culturesupernatant of stably expressing cells was quantified by an anti-humanIgG ELISA kit (Bethyl Laboratories, Inc.). In contrast with thecombination of only HC1 and LC1 (12.93 pg/cell/day), or the combinationof HC2 and LC2 (14.02 pg/cell/day) that is poor in activity of forming atetramer, significantly high (37.45 pg/cell/day) antibody production wasobserved when four genes, HC1, LC1, HC2, and LC2 were installed. Thissuggests that the bispecific antibody is produced efficiently. Thisexpression level is comparable to the gene expression level in a generalcell strain established by CHO cells using gene amplification (about 90pg/cell/day at maximum) (Non-Patent Document 88). This suggests that asa method for stably producing a bispecific antibody for which a stablyexpressing cell strain was been difficult to be obtained by conventionalmethods, the stealth RNA vector is very useful. The total nucleotidelength of the genes used in the present Example is 6.7 k nucleotides,and this size cannot be realized by a method using a conventional RNAvector.

INDUSTRIAL APPLICABILITY

The present invention is useful in various industrial fields includingreprogramming of human cells including preparation of inducedpluripotent stem cells (iPS cells), production of protein drugs, genetherapy by various genes including giant genes, and expression ofdrug-discovery target molecules.

1. A stealth RNA gene expression system comprising: a negative-sensesingle-stranded RNA (A) having RNA sequences (1) to (8) below, asingle-stranded RNA binding protein (B), and an RNA-dependent RNApolymerase (C), wherein the stealth RNA gene expression system is acomplex that does not activate an innate immune system: (1) target RNAsequences encoding any given protein or functional RNA, (2) RNAsequences constituting noncoding region(s) and derived from mRNA(s)expressed in animal cells, (3) transcription start signal sequencesrecognized by the RNA-dependent RNA polymerase, (4) transcriptiontermination signal sequences recognized by the polymerase enzyme, (5)RNA sequences containing replication origins recognized by thepolymerase enzyme, (6) RNA sequences encoding the polymerase enzyme withcodons optimized for a biological species from which cells fortransfection are derived, (7) an RNA sequence encoding a protein thatregulates activity of the polymerase enzyme with codons optimized for abiological species from which cells for transfection are derived, and(8) au RNA sequence encoding the single-stranded RNA binding proteinwith codons optimized for a biological species from which cells fortransfection are derived.
 2. The stealth RNA gene expression systemaccording to claim 1, wherein the target RNA sequences of the (1)contain at least six genes, or are RNA sequences having a total lengthof 5000 or more nucleotides.
 3. The stealth RNA gene expression systemaccording to claim 1, wherein the RNA sequences of the (2) are RNAsequences each having a length of 5 to 49 nucleotides derived from mRNAof human gene(s).
 4. The stealth RNA gene expression system accordingclaim 1, wherein each of the RNA sequences of the (2) having sequencesidentical to or different from one another is placed adjacent to 3′terminal site and/or 5′ terminal site of each of gene sequencescontained in the target RNA sequences, of the (1).
 5. The stealth RNAgene expression system according to claim 1, wherein the RNA-dependentRNA polymerase encoded by the RNA sequences of the (6) consists of Lprotein and P protein derived from an RNA virus belonging to aparamyxovirus family, the protein that regulates activity of thepolymerase enzyme encoded by the RNA sequence of the (7) is C proteinderived from the same virus as the RNA virus, the single-stranded RNAbinding protein encoded by the RNA sequence of the (8) is NP proteinderived from the same virus as the RNA virus, and all of the RNAsequences of the (3) to (5) are RNA sequences containing a transcriptionstart signal, a transcription termination signal, or a replicationorigin sequence derived from a genome of the same virus as the RNAvirus.
 6. The stealth RNA gene expression system according to claim 5,wherein the RNA sequences encoding the L protein, P protein, C proteinand NP protein are optimized for human cells, and have a GC contentadjusted within a range of 50 to 60%.
 7. The stealth RNA gene expressionsystem according to claim 6, wherein the RNA virus belonging to aparamyxovirus family is an RNA virus selected from the group consistingof Sendai virus, human parainfluenza virus, and Newcastle disease virus.8. The stealth RNA gene expression system according to claim 1, whereinthe transcription start signal sequences of the (3) are RNA sequencesselected from the group of RNA sequences consisting of 3′-UCCCACUUUC-5′(SEQ ID NO: 1), 3′-UCCCUAUUUC-5′ (SEQ ID NO: 2), 3′-UCCCACUUAC-5′ (SEQID NO: 3), 3′-UCCUAAUUUC-5′ (SEQ ID NO: 7), and 3′-UGCCCAUCUUC-5′ (SEQID NO: 9), and the transcription termination signal sequences of the (4)are RNA sequences selected from the group of RNA sequences consisting of3′-AAUUCUUUUU-5′ (SEQ ID NO: 4), 3′-CAUUCUUUUU-5′ (SEQ ID NO: 5),3′-UAUUCUUUUU-5′ (SEQ ID NO: 6), and 3′-UUAUUCUUUUU-5′ (SEQ ID NO: 8).9. The stealth RNA gene expression system according to claim 4, whereineach of the transcription start signal sequences of the (3) havingsequence identical to or different from one another is placed adjacentto 3′ terminal site of each of the RNA sequences of the (2) that isplaced adjacent to 3′ terminal site of each of gene sequences containedin the target RNA sequences of the (1), and each of the transcriptiontermination signal sequences of the (4) is placed adjacent to 5′terminal site of the RNA sequence that is placed adjacent to 5′ terminalsite of each of gene sequences contained in the target RNA sequences ofthe (1).
 10. The stealth RNA gene expression system according to claim7, wherein the RNA sequences containing replication origins of the (5)contain the following sequences: (a) an RNA sequence represented by3′-UGGUCUGUUCUC-5′ (SEQ ID NO: 11) or 3′-UGGUUUGUUCUC-5′ (SEQ ID NO:12), (b) an RNA sequence represented by 3′-GAGAACAGACCA-5′ (SEQ ID NO:13) or 3′-GAGAACAAACCA-5′ (SEQ ID NO: 14), (c) an RNA sequencerepresented by 3′-(CNNNNN)₃-5′ (SEQ ID NO: 15), and (d) an RNA sequencerepresented by 3′-(NNNNNG)₃-5′ (SEQ ID NO: 16).
 11. The stealth RNA geneexpression system according to claim 10, wherein the RNA sequence of the(a) is positioned at the 3′ terminus of the negative-sensesingle-stranded RNA (A), and the RNA sequence of the (b) is positionedat the 5′ terminus.
 12. The stealth RNA gene expression system accordingto claim 10, wherein the RNA sequence of the (c) starts at 79thnucleotide from the 3′ terminus of the negative-sense single-strandedRNA (A), and the RNA sequence of the (d) starts at 96th nucleotide from5′ terminus.
 13. The stealth RNA gene expression system according toclaim 10, wherein the RNA sequences containing replication origins ofthe (5) further contain in a position of 97th to 116th nucleotides fromthe 3′ terminus of the negative-sense single-stranded RNA (A), an RNAsequence of (e) 3′-AAAGAAACGACGGUUUCA-5′ (SEQ ID NO: 17) or an RNAsequence having the same length of 18 nucleotides as the (e).
 14. Astealth RNA vector comprising a complex composed of the stealth RNA geneexpression system according to claim 1, and having activity ofintroducing the complex into animal cells, that does not activate aninnate immune system.
 15. The stealth RNA vector according to claim 14,that forms a virus particle having ability to infect animal cells. 16.An animal cell transfected with the stealth RNA vector according toclaim
 14. 17. A stealth RNA which is a negative-sense single-strandedRNA (A) having RNA sequences of (1) to (8) below, capable of forming acomplex that does not activate an innate immune system together with asingle-stranded RNA binding protein (B), and an RNA-dependent RNApolymerase (c): (1) target RNA sequences encoding any given protein orfunctional RNA, (2) RNA sequences constituting noncoding region(s) thatare unrecognizable by an innate immune system, (3) transcription startsignal sequences recognized by an RNA-dependent RNA polymerase, (4)transcription termination signal sequences recognized by the polymeraseenzyme, (5) RNA sequences containing replication origins recognized bythe polymerase enzyme, (6) RNA sequences encoding the polymerase enzymeand having a structure optimized to be unrecognizable by an innateimmune system, (7) an RNA sequence encoding a protein that regulatesactivity of the polymerase enzyme, and having a structure optimized tobe unrecognizable by an innate immune system, and (8) an RNA sequenceencoding a single-stranded RNA binding protein and having a structureoptimized to be unrecognizable by an innate immune system.
 18. Thestealth RNA according to claim 17, wherein RNA sequences containingreplication origins recognized by the RNA-dependent RNA polymerase ofthe (5) are located at the 3′ terminal site and the 5′ terminal site ofthe negative-sense single-stranded RNA (A), and the RNA sequence locatedat the 3′ terminal site and the RNA sequence located at the 5′ terminalsite include RNA sequences complementary to each other.
 19. The stealthRNA according to claim 17, wherein each of the transcription startsignal sequences of the (3) having sequences identical to or differentfrom one another is placed adjacent to 3′ terminal site of each of theRNA sequences of the (2) that is placed adjacent to 3′ terminal site ofeach of plural gene sequences contained in the target RNA sequences ofthe (1), and each of the transcription termination signal sequences ofthe (4) is placed adjacent to further 5′ terminal site of the RNAsequence that is placed adjacent to 5′ terminal site of each of pluralgene sequences contained in the target RNA sequence of the (1).
 20. Thestealth RNA according to claim 17, wherein each of the transcriptionstart signal sequences of the (3) having sequences identical to ordifferent from one another is placed adjacent to 3′ terminal site ofeach of the RNA sequences of the (2) that is placed adjacent to 3′terminal site of each of plural gene sequences contained in the targetRNA sequences of the (1); each of the transcription termination signalsequences of the (4) is placed adjacent to 5′ terminal site of the RNAsequence that is placed adjacent to 5′ terminal site of each of pluralgene sequences contained in the target RNA sequences of the (1); andboth of them constitute a cassette structure together with restrictionsites located at both ends of the cassette that can be cleaved by pluralrestriction endonucleases, and plural cassette structures are bound toeach other.
 21. A method for reconstituting a stealth gene expressionsystem, comprising the following processes (1) to (5): (1) preparing anEscherichia coli expressing T7 RNA polymerase, (2) introducing into theEscherichia coli host of the (1), at least a vector for Escherichia colicarrying an RNA encoding an RNA-dependent RNA polymerase and an RNAbinding protein, and a vector for Escherichia coli for expressing a DNAencoding RNA binding protein, together with the negative-sensesingle-stranded RNA (A) according to any one of claims 1 to 13 totransform the host, (3) forming a complex of the negative-sensesingle-stranded RNA containing exogenous gene RNA expressed by T7 RNApolymerase, and RNA binding protein in the transformed Escherichia coliof the (2), (4) preparing animal cells in which an RNA-dependent RNApolymerase is expressed, and (5) introducing the complex of thenegative-sense single-stranded RNA and the RNA binding protein obtainedin the (3) into an animal cell host of the (4) to reconstitute a stealthRNA gene expression system composed of the negative-sensesingle-stranded RNA, and the complex of the RNA binding protein and theRNA-dependent RNA polymerase.
 22. A DNA-based tandem cassette having twocloning sites A and B, the tandem cassette being composed of (1)multimerization site A, (2) transcription start signal A, (3) noncodingsequence A1, (4) cloning site A, (5) noncoding region A2, (6)transcription termination signal A, (7) transcription start signal B,(8) noncoding sequence B1, (9) cloning site B, (10) noncoding region B2,(11) transcription termination signal B, and (12) multimerization site Bin order from the 5′ terminus, the multimerization site A of the (1),and multimerization site B of the (12) being DNAs that are identical toor different from each other and each containing a recognition site byrestriction endonuclease and/or a recognition site by site-specificrecombinase, the transcription start signal A of the (2), andtranscription start signal B of the (7) being DNAs that are identical toor different from each other and each containing a transcription startsignal recognized by the RNA-dependent RNA polymerase when transcribedto RNA, the noncoding sequence A1 of the (1), noncoding region A2 of the(5), noncoding sequence B of the (8), and noncoding region B2 of the(10) being DNAs that are identical to or different from one another andeach becoming RNA that is not recognized by an innate immune system of ahost cell when transcribed to RNA, the cloning site A of the (4), andcloning site B of the (9) being DNAs that are identical to or differentfrom each other and each containing one or more recognition sites byrestriction endonuclease and/or recognition sites by site-specificrecombinase, the transcription termination signal A of the (6), andtranscription termination signal B of the (11) being DNAs that areidentical to or different from each other and each containing atranscription termination signal recognized by the RNA-dependent RNApolymerase when transcribed to RNA.
 23. The tandem cassette according toclaim 22, wherein the cloning site A of the (4) contains a recognitionsite by restriction endonuclease A, and a recognition site byrestriction endonuclease C in order from 5′ terminal side, and thecloning site B of the (9) contains a recognition site by restrictionendonuclease D, and a recognition site by restriction endonuclease B inorder from 5′ terminal side, provided that the restriction endonucleaseA and the restriction endonuclease D give single-stranded protrudingends of the same order, and the restriction endonuclease C and therestriction endonuclease B give single-stranded protruding ends of thesame sequence.
 24. The tandem cassette according to claim 22, whereinboth of the multimerization site A of the (1), and multimerization siteB of the (12) are DNAs containing a recognition site by a restrictionendonuclease giving a single-stranded protruding end of any sequencerepresented by NN or NNN.
 25. The tandem cassette according to claim 22,wherein the noncoding sequence A1 of the (3), noncoding region A2 of the(5), noncoding sequence B1 of the (8), and noncoding region B2 of the(10) are identical to or different from one another and each of them iscDNA corresponding to a partial sequence of RNA sequences derived frommRNA expressed in animal cells, and one of human-derived genes identicalto or different from each other is inserted into the cloning site A ofthe (4), and cloning site B of the (9).